Crosstalk between salicylic acid signalling and the circadian clock promotes an effective immune response in plants
Introduction
The rotation of our planet creates 24-h cycles in our environments, such as in light levels and temperature. To anticipate these rhythms, the circadian clock has evolved as an internal timekeeping mechanism in almost all eukaryotes. It synchronizes an organism’s behavioural and physiological processes with the rhythmic changes in its surroundings1,2. The temporal separation or coordination of process that results from this timekeeping enables the optimization of resource use in an organism’s everyday function3,4.
As sessile organisms, plants cannot escape unfavourable conditions, evade herbivores, or move to find more distant resources. Therefore, the main challenge for plants is the effective use of available resources to improve fitness. Plants utilize the circadian clock to track and anticipate periodic environmental changes and stressors. This results in the prioritizing of distinct biological responses at different times of day or year according to the anticipated environmental conditions4,5,6,7.
In Arabidopsis, circadian rhythms involve an interconnected network of transcriptional/translational feedback loops, in which circadian components activate or repress each other’s activity in a cyclical manner7. The core of the circadian clock consist of the morning expressed MYB-related transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and the evening expressed TIMING OF CAB EXPRESSION 1 (TOC1). CCA1, LHY and TOC1 negatively regulate each other’s expression alongside other components of the clock8,9,10,11,12. The rhythmic oscillations of CCA1, LHY and TOC1 regulate many downstream pathways through periodic gene expression. An estimated 30% of the Arabidopsis thaliana (Arabidopsis) transcriptome is under circadian control13. This includes genes involved in regulating flowering, biomass, photosynthesis, water use, temperature stress responses and pathogen defences4,14,15.
One of the downstream pathways that the clock modulates is the immune system16. Plant pathogens and pests cause devastating losses to food crops worldwide17. Understanding the mechanisms that underpin plant immunity is critical to improving crop productivity and to meet the exponentially increasing food demand. Pathogen recognition is conferred through two immune pathways: PAMP triggered immunity (PTI) and effector triggered immunity (ETI)18,19. Pathogen recognition by these interconnected pathways leads to accumulation of the immune hormone salicylic acid (SA) that activates the expression of thousands of immune genes, many via the activation of the master transcriptional coactivator, NPR120,21. NPR1 is responsible for the regulation of pathogenesis related (PR) genes including PR1. In addition to activating local immune responses, SA and NPR1 are also required for the onset of systemic acquired resistance (SAR), which provides long-lasting protection throughout the entire plant against a broad spectrum of pathogens22,23,24,25.
Basal levels of SA are under circadian control with SA abundance peaking in the middle of the night, which is thought to prime the immune response for dawn, when plants are most vulnerable to pathogen infection26,27,28. In accordance, Arabidopsis displays enhanced resistance in the subjective morning and increased susceptibility at subjective midnight29. This disparate resistance is lost in plants overexpressing CCA1 (CCA1ox) and in elf3-1 mutants, both of which are arrhythmic at the gene expression levels. Furthermore, key immune genes are under circadian regulation, including the pathogen recognition receptors FLS2 and EFR, as well as components of the SA biosynthesis, ICS1 and EDS129. Interestingly, rhythmic levels ofis an SA receptor and master regulator the active NPR1 monomer have also been observed30.
There is also evidence for reciprocal modulation of the circadian clock by the immune system, however the nature of clock alteration and the extent to which it is modified are unclear. Arabidopsis infected with Pseudomonas or treated with the bacterial elicitor flg22 display a significantly shortened circadian period31. The current research is not in agreement regarding the effect of SA on circadian clock rhythms. Zhou et al.30 found that SA treatment caused an increase in the amplitude of TOC1 but not CCA1 rhythms in an NPR1-dependent manner. On the other hand, SA treatment has also been reported to cause a significant reduction of amplitude and delay in circadian phase32. Furthermore, Philippou et al.33 indicate that SA treatment shortens the period of CCA1 and TOC1 rhythms in the absence of sucrose.
Taken together, these findings point to extensive crosstalk between the immune system and the circadian clock. However, the exact mechanisms and extent of cross modulation are poorly understood.
In this study, we further explore the relationship between SA-dependent immune signalling and the circadian clock in Arabidopsis. We aimed to establish the role of NPR1 in the link between SA-signalling and circadian clock rhythms and investigate the potential involvement of core clock genes CCA1 and TOC1 in the modulation between the two systems. Indeed, our study reveals the critical role of NPR1 in the SA-induced modulation of CCA1 and TOC1 rhythms. We also establish the importance of clock gene CCA1 in launching an effective SA-induced immune response, demonstrated in both SA-induced PR1 transcript levels and SA-induced resistance to Pseudomonas.
Results
SA treatment shortens the period of the circadian clock
Conflicting reports exist on the effects of SA on the circadian clock in plants. Therefore, we first aimed to consolidate the effect of SA on the clock within our experimental systems and conditions. Leaf discs of plants expressing firefly luciferase (LUC) driven by the rhythmic promoters of either CCA1 or TOC1 (CCA1pro:LUC and TOC1pro:LUC, respectively) were subjected to a range of SA concentrations.
Treatments with 100 µM SA (Supplementary Fig. 2) or 1 mM SA (Fig. 1 and Supplementary Fig. 1) shortened the periods of both CCA1 and TOC1 under constant light conditions. Higher concentrations of SA abolished rhythms while lower concentrations did not significantly alter rhythms (Supplementary Fig. 2). The raw luminescence data indicate that, though the period-shortening effect is consistent, there is not a consistent effect of 1 mM SA treatment on amplitude (Supplementary Fig. 1).
Promoter activity of CCA1 (a, b) and TOC1 (c, d) was observed by measuring luminescence in CCA1pro:LUC and TOC1pro:LUC leaf disks, respectively. Leaf disks were treated with 1 mM SA (blue) or mock treated with water (grey). The mean period (b, d) was calculated from the 24–120 hr time window. An unpaired t test was performed between each mock and treated data set for CCA1 and TOC1: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t-test. Error bars indicate mean ± SEM (n = 7). Data are from a single experiment representative of 3 independent repeats.
Thus far, we have observed the effects of continuous SA treatment on clock rhythms, however, as pathogen-induced SA accumulation is transient34, we next investigated the effect of transient SA treatment on CCA1 rhythms. CCA1pro:LUC leaf disks were treated with SA at 5 different time points over 24 h. The media was replaced with SA-free imaging media 8 h post-treatment. Only time points LL34 and LL44 exhibited significant period shortening (Supplementary Fig. 3). We observed no effect on phase or amplitude.
Overall, these results indicate that exogenous SA treatment modulates the circadian clock in a dose-dependent manner.
SA-induced period shortening is dependent on NPR1
Our results suggest that SA treatment alters circadian clock rhythms in Arabidopsis. NPR1 is an SA receptor and master regulator of SA-responsive immune gene expression20. Zhou et al.30 previously reported that the effect of SA on TOC1 is dependent on NPR1. Contrasting results reported by Li et al.32 argue that NPR1 antagonizes the clock response to SA treatment. As NPR1 is a regulator of SA-dependent immunity, it may mediate feedback between SA and the clock. However, in previous studies that observed circadian gene expression of NPR1 mutants, no effect on period was found30,32.
To investigate this, we observed CCA1 and TOC1 rhythms in npr1-1 mutants. We crossed CCA1pro:LUC and TOC1pro:LUC with npr1-1 mutants. First, we compared the rhythms of CCA1 and TOC1 in wild-type (WT) versus npr1-1 mutant background (Fig. 2). We found that npr1-1 mutants had a significantly longer period as observed for both CCA1 (Fig. 2a, b) and TOC1 (Fig. 2c, d). Although the period lengthening effect is small, the detailed time resolution provided by luciferase reporters allowed us to evidence a clear effect of NPR1 on circadian gene expression in uninduced conditions
Promoter activity of CCA1 (a, b) or TOC1 (c, d) was measured in the parent line parent line (grey) and the npr1-1 mutant (orange). CCA1 promoter activity (a, b) was visualized by measuring luminescence of CCA1pro:LUC in wild-type (grey) versus CCA1pro:LUC in npr1-1 (orange) leaf disks. TOC1 promoter activity (c, d) was visualized by measuring luminescence of TOC1pro:LUC in wild-type (grey) versus TOC1pro:LUC in npr1-1 (orange) leaf disks. The mean period was calculated from the 24–120 h time window from traces of CCA1 (b) and TOC1 (d): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t test. Error bars represent mean ± SEM (n = 6). The data shown here are from a single experiment representative of 3 independent repeats.
To test if NPR1 also mediates the effects of SA on circadian period shortening (Fig. 1), we subjected our clock marker lines in the WT and npr1-1 backgrounds to SA treatment. As opposed to the WT background (Fig. 1), SA treatment of CCA1pro:LUC in npr1-1 and TOC1pro:LUC in npr1-1 leaf discs did not alter rhythms (Fig. 3). Combined, these data demonstrate that NPR1 affects timekeeping and that the effect of SA on the circadian clock is dependent on NPR1. Thus, basal and SA-induced NPR1 are part of the input system of the plant circadian clock.
Promoter activity of CCA1 (a, b) or TOC1 (c, d) was observed by measuring luminescence in CCA1pro:LUC in npr1-1 and TOC1pro:LUC in npr1-1 leaf disks, respectively. Leaf disks were treated with 1 mM SA (blue) or mock treated with water (orange). The mean period was calculated from the 24–120 h time window from traces of CCA1 (b) or TOC1 (d): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t test. Error bars indicate mean ± SEM (n = 6). The data shown here are from a single experiment representative of 2 independent repeats.
SA-induced PR1 transcript levels are gated by the circadian clock
Thus far, NPR1 appears to be one of the key elements in crosstalk between SA levels and the circadian clock. NPR1 is a master regulator of SA-responsive gene expression, including the antimicrobial gene PATHOGENESIS RELATED 1 (PR1). As endogenous basal levels of SA as well as the abundance of monomeric NPR1 protein exhibit circadian oscillations28,30, we investigated if PR1 transcript levels are also rhythmic. We measured PR1 transcript levels in wild-type plants by sampling every 3 h for 2 days. In the absence of an immune inducer, basal PR1 transcript levels remained very low and did not exhibit rhythmicity (Supplementary Fig. 4). However, when we subjected plants to SA at 3-h intervals, we found that SA-induced PR1 transcript levels were consistently higher during the subjective night than the subjective day (Fig. 4). Rhythmicity analysis of the mean SA-induced PR1 transcript level of both biological replicates combined was assessed using the eJTK algorithm35. This analysis revealed that SA-induced PR1 transcript levels are not rhythmic (p < 0.05) Thus, the circadian clock appears to gate SA-induced PR1 transcript levels, though it is not rhythmic.
Col-0 seedlings were grown on MS plates under 12/12 light/dark conditions for 14 days before being released into constant light (LL). Every 3 h, plates were sprayed with 1 mM SA. Relative transcript levels of PR1 were measured 6 h post-treatment. The data points are the mean ± SEM (n = 3) of quantitative PCR results from two biological replicates (light blue versus dark blue). The black line represents the mean values of the two biological replicates.
To investigate this further, we compared SA-induced PR1 transcript levels at subjective dawn versus dusk in wild-type as well as in arrhythmic CCA1ox plants36 and cca1lhy mutants that exhibit a shortened period31. As expected, wild-type plants exhibited stronger induction of PR1 transcript levels in response to SA treatment at dusk (Fig. 5a, d, g). The difference in SA-induced PR1 transcript levels between dusk and dawn was increased in CCA1ox plants (Fig. 5b, e, h), but completely lost in the cca1lhy mutant (Fig. 5c, f, i). These data indicate that core clock component CCA1 gates SA-dependent transcript levels of PR1 and enhances responsiveness to SA at dusk.
Col-0 (a, d, g), CCA1ox (b, e, h) and cca1lhy (c, f, i) seedlings were sprayed with 1 mM SA or mock (water) at subjective dawn versus dusk. Seedlings were sampled 6 h after treatment and PR1transcript levels were measured. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t test. Error bars indicate mean ± SEM (n = 4). The data shown here are from three independent experiments: experiment 1 (a–c); experiment 2 (d–f); experiment 3 (g–i). We present all the results to demonstrate consistency between the independent repeats.
CCA1 is required for SA-induced resistance
Our results to this point demonstrate evidence of crosstalk between SA signalling and the circadian clock. In spite of the evidence for clock regulation of SA levels and immune gene expression27,28,29, no direct evidence exists for circadian involvement in the process of SA-induced resistance to pathogens. Therefore, we investigated the role of the circadian clock in establishing SA-induced resistance. We sprayed wild-type (Col-0), CCA1ox, cca1lhy, TOC1ox (arrhythmic37) and toc1-101 (short period38) plants with SA at subjective dawn to mimic pathogen infection, followed 24 h later by infiltration with Pseudomonas syringae pv. maculicola (Psm) ES4326. We have displayed all the results for this assay (Fig. 6 and Supplementary Fig. 5) as the quantification of bacterial growth can be highly variable. As expected, wild-type plants displayed a significantly lower level of infection after treatment with SA (Fig. 6 and Supplementary Fig. 5). TOC1ox plants and toc1-101 mutants also displayed SA-induced resistance, indicating that neither TOC1 expression nor its rhythmicity is required for SA-mediated resistance. However, SA-induced resistance was not observed in CCA1ox plants or in cca1lhy mutants. These results indicate that both CCA1 expression as well as its rhythmic expression are required to establish SA-dependent disease resistance. These results further evidence the critical role for CCA1 in linking the circadian clock, SA signalling, and immunity.
Col-0, CCA1ox, cca1lhy, TOC1ox and toc1-101 plants were grown under 12/12 light/dark conditions for 3 weeks. At dawn on day 21 (LL0), the plants were released into constant light conditions. At LL24 the plants were sprayed with 1 mM SA or mock (water). At LL48 the plants were infected with Pseudomonas syringae pv. maculicola (Psm). The level of bacterial growth was measured 3 days post infection. a Leaves representative of the visual median level of infection at 3 days post-infection for each genotype and treatment. b The infection level at 3 days post-infection expressed by colony forming units (CFU, y-axis is logarithmic) in leaf disk extracts. Error bars represent mean ± SEM (n = 8 individual leaves). A two-way ANOVA and Tukey’s HSD Test were preformed to analyse the differences in bacterial growth between treatments and genotypes. The letters indicate significant differences between groups (p < 0.05). The data shown here are from a single experiment, representative of 3 independent experiments (Col-0 versus CCA1ox, cca1lhy or TOC1ox [Supplementary Fig. 5a–c]) or 2 independent experiments (Col-0 versus toc1-101 [Supplementary Fig. 5c, d]).
Discussion
Plant pathogens are responsible for large reductions in agricultural productivity17. A functioning circadian clock has been shown to enhance resistance to pests and pathogens both pre- and post-harvest27,39. The regulation of the immune system by the circadian clock has long been theorized to prime the immune system to the time of day that pathogen attack is most likely, thereby conserving the plants resources27. The alteration of circadian rhythms during infection may function to prioritize resource allocation towards an immune response by resetting the circadian clock to a time of day that facilitates this through its regulation of other plant processes.
The results presented in this study indicate that the immune hormone SA modulates that circadian clock, inducing NPR1-dependent period shortening. Previous studies have reported conflicting results regarding the effect of SA on circadian clock rhythms. Zhang et al.31 found that treating seedlings with the SA mimic benzo(1,2,3)thiadiazole-7-carbothioic acid (BTH) had no effect on CCA1 promoter rhythms. In a similar study, Zhou et al.30 found that treatment with SA increased the amplitude of TOC1 rhythms but not CCA1 rhythms in 3-week-old Arabidopsis.
Li et al.32 monitored the effect of transient (4 h) SA treatment on clock gene rhythms, observing a significant reduction in amplitude and a delay in phase for CCA1, TOC1 and LHY rhythms. This delay in phase was larger when SA treatment began closer to subjective dawn, though no such pattern was observed in the amplitude reduction. In contrast, when we applied SA transiently (8 h), period shortening of CCA1 rhythms occurred at LL34 and LL44, but no effect on phase or amplitude was observed.
None of the studies mentioned above observed any effect of long-term SA exposure on period. However, Philippou et al.33 observed SA-induced period shortening of GI (GIGANTEA), CCA1, CCR2 and TOC1 rhythms, which was attenuated by the addition of sucrose. Therefore, the discrepancies between our study and the previous studies following SA treatment could be due to differences in the state of the plant material when monitored, such as the presence of sucrose, whole plant versus leaf disk or potentially light levels. Overall, we contribute to the conclusion that there is dose-dependent feedback between the immune system and the circadian clock via SA signalling.
Li et al.32 found that the npr1-1 mutant displayed a larger reduction in the amplitude of CCA1 and LHY upon SA treatment, suggesting that NPR1 acts as an antagonist of the effects of SA on the clock. Zhou et al.30 also observed that NPR1 was essential for the effect of SA on the clock as the npr1-1 mutant did not exhibit the SA-induced increase in amplitude of TOC1 rhythms. We found that the SA receptor NPR1 was crucial for the SA-induced period shortening. The amplitude effects we observed were not consistent between replicates and experiments, and therefore we chose not to report on them here. Significantly, we also found that the npr1-1 mutant displayed a longer period for both CCA1 and TOC1 expression in uninduced conditions. This novel finding indicates that basal NPR1 levels, as well as SA-induced NPR1, modulates the circadian clock. The NPR1-dependent SA alteration of circadian clock gene rhythms may function to harness that clock’s modulation of other cellular processes in order to prioritize an immune response when pathogen attack is detected.
Existing literature reports the importance of CCA1 in launching an immune response to primary bacterial infection. Zhang et al.31 report that CCA1ox plants exhibit increased susceptibility to Pseudomonas infection compared to wild type. Conversely, it has also been observed that CCA1 overexpression confers increased resistance to oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) while the cca1 mutant displayed increased susceptibility31,40.
Our study aimed to expand upon this knowledge of CCA1’s role in immunity by investigating the potential involvement of core clock genes in SA-induced immunity. We demonstrate the importance of the circadian clock in launching an effective immune response and expand upon the critical role of CCA1 in this coordination. We found that SA-induced PR1 transcript levels were higher during the subjective night, though not rhythmic. Plants overexpressing CCA1 exhibited a larger difference in SA-induced PR1 transcript levels between dawn and dusk compared to wild type. Conversely, plants lacking CCA1 did not display the temporal difference in SA-induced PR1 transcript levels. This suggests that clock gene CCA1 is required for the temporal difference in SA-induced PR1 transcript levels. However, as cca1lhy exhibits a shorter period under constant conditions31,41, it is possible that we missed the peak/trough in SA-induced PR1 transcript levels at the time points used in our experiment.
In addition, we observed that CCA1 is also crucial for the downstream function of establishing SA-induced resistance to Pseudomonas. We found that priming with SA did not induce resistance in CCA1ox or cca1lhy as it did in Col-0, suggesting that CCA1 is crucial for the resistance response following SA treatment. We did not observe increased susceptibility to Pseudomonas in CCA1ox as reported by Zhang et al.31. CCA1 is a key morning clock gene and master regulator of circadian rhythms. Plants overexpressing CCA1 have an arrhythmic clock under LD and LL conditions36. TOC1ox (also arrhythmic37) displayed SA-induced resistance to Pseudomonas, suggesting that the lack of SA-induced resistance in CCA1ox is not due to overall clock arrhythmia. As the cca1lhy mutant also did not display an SA-induced response, regulated CCA1 expression levels appear to be important for establishing SA-induced resistance. We did not observe increased sensitivity to SA treatment in toc1-101 mutant plants compared to Col-0 as reported by Zhou et al.30. This could be due to differences in the timing of treatment and inoculation, which were carried out at subjective dusk (ZT12) by Zhou et al.30 but at subjective dawn (ZT0) in our experiments. Overall, these findings indicate that the function of CCA1 as transcription factor outside of its role as a core clock component is responsible for its effect on the SA-induced immune response.
From our study, it is evident that SA utilizes NPR1 for modulating rhythmicity of core clock components. NPR1 is crucial for many immune processes downstream of SA. The npr1-1 mutant displays increased susceptibility to infection, a lack of PR gene expression upon infection/SA treatment and an inability to establish resistance following priming with SA20,21. As we have shown that vice versa the circadian clock is also involved in regulating SA induced PR gene expression and resistance to Pseudomonas, it would be interesting to investigate whether NPR1 plays a role in this interaction, potentially facilitating crosstalk between the circadian clock and the immune system in both directions. Additionally, as redox regulation of NPR1 conformation and localization is a key step in its activation42,43, daily redox rhythms44,45 may control the interplay between the circadian clock and NPR1.
Our study provides evidence of the reciprocal regulation between the circadian clock and SA signalling. We show the importance of NPR1 in this interaction and that clock gene CCA1 is key for regulation of SA-induced immune responses. The findings presented here further our understanding of the interactions between the circadian clock and the immune system and how they work together to produce an effective immune response. Continued study of the systems controlling plant immunity is essential to prioritize plant health and thus food security.
Methods
Plant materials and growth conditions
Arabidopsis thaliana were grown on soil for 3-4 weeks, unless otherwise stated, in growth cabinets (Percival Scientific Inc.) set at constant 21 °C with a light intensity of 70-100 µmoles m-2 s-1 (Fusion 18 W T8 2 ft Triphosphor Fluorescent Tube 4000 K) under 12 h light/12 h dark. All wild type and mutant plants were from the Col-0 genetic background and were described previously: npr1-121; CCA1ox36; TOC1ox37; cca1lhy31; toc1-10138; CCA1pro:LUC46 and TOC1pro:LUC47. Experiments involving NPR1 use the single mutant allele npr1-1, as the presence of second-site mutation is unlikely due to extensive backcrossing20,21 and whole genome sequencing48.
CCA1pro:LUC npr1-1 and TOC1pro:LUC npr1-1 plants were made by crossing CCA1pro:LUC with npr1-1 and TOC1pro:LUC with npr1-1 respectively. To obtain homozygous lines of npr1-1 plants, individuals in the F1 and F2 generations were evaluated by polymerase chain reaction (PCR) genotyping and restriction fragment length polymorphism (RFLP) analysis. DNA was extracted using a one-step protocol previously described in Edwards et al.49. For PCR amplification, 0.5 μl of the DNA extract was added to 10 μl of 5X GoTaq Reaction Buffer (Promega) and 1 μl of each primer (10 μM, NPR1 F: CTCGAATGTACATAAGGC, NPR1 R: CGGTTCTACCTTCCAAAG20), to a total volume of 20 μl. PCR conditions were 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, followed by 72 °C for 5 min. PCR products were digested with 0.4 μl of restriction enzyme NlaIII for 2 h at 37 °C (New England Biolabs). Digested PCR products were run on 2% agarose gels with SYBR Safe (Invitrogen) at 100 V and were imaged using the Odyssey FC imaging system (LI-COR).
Homozygous npr1-1 lines were tested for homozygosity of the luciferase transgene (either CCA1pro:LUC or TOC1pro:LUC) in F3. A minimum of 50 seeds of each line were visualized for luminescence by sowing one seed per well, on top of 200 μl 0.5 MS in black 96-well plates (Greiner bio-one, 655075). Seedlings were grown for 10 days in an incubator (Sanyo) under long-day conditions. 20 μl of 1 mM D-luciferin (Biosynth AG) was added to each well and luminescence was visualized using the ALLIGATOR luminescence imaging system by exposing the camera for 8 min. Ratios of luminescence were assessed: homozygous lines which displayed luminescence in all seedlings were used for subsequent experiments.
Bioluminescence assay of leaf discs
CCA1pro:LUC, TOC1pro:LUC, CCA1pro:LUC npr1-1 and TOC1pro:LUC npr1-1 were grown on soil under LD conditions (see plant materials and methods). At 3-4 weeks old, leaf disks were taken from leaves 5-6 and placed in the wells of a white, flat-bottom 96-well plates (Greiner bio-one, 655075) with the adaxial side up. Each well also contained 200 µl of filter sterilized imaging solution: 0.5 MS pH 7.5, 50 µg/ml ampicillin, 1.5 mM D-luciferin and SA/H2O to the desired final concentration. The plate was sealed with a clear, gas permeable lid (4titude, 4ti-0516/96). Luminescence was measured by a LB942 Tristar2 plate reader (Berthold Technologies Ltd) every 50 min for 3 s per well. Leaf disks were kept under continuous red (630 nm) and blue (470 nm) LED light at 17.5 µmoles m−2 s−1 each at 20–21 °C. Results were analysed using GraphPad Prism and Biodare250 and period was estimated using the Fast Fourier Transform-Non-Linear Least Squares (FFT-NLLS) function. Unless otherwise specified, luminescence data are displayed as residual luminescence, wherein a polynomial was fitted to the raw luminescence data and the residual calculated from this using GraphPad Prism.
SA treatment for quantitative PCR in wildtype over 48 h
Wild-type plants (Col-0) were grown on MS agar plates under 12 h light/12 h dark conditions at 21 °C with a light intensity of 70–100 µmoles m−2 s−1 (Fusion 18 W T8 2 ft Triphosphor Fluorescent Tube 4000 K). After 14 days, the seedlings were released into constant light (LL). Plates were removed from the growth cabinet and sprayed with 1 mM SA or mock (H2O) at 3-h intervals beginning at LL24 and finishing at LL72. After spraying, the plates were put into separate growth cabinets from the untreated plates. The seedlings were harvested and frozen in liquid nitrogen 6 h after treatment.
SA treatment for quantitative PCR in clock mutants
Wild type (Col-0), CCA1ox and cca1lhy plants were grown on MS agar plates under 12 h light/12 h dark conditions at 21 °C with a light intensity of 70–100 µmoles m−2 s−1 (Fusion 18 W T8 2 ft Triphosphor Fluorescent Tube 4000 K). Plates were placed under split entrainment conditions in separate growth cabinets, aligning the time of dawn and dusk between the two cabinets. After 14 days, the seedlings were released into LL. Plates were sprayed with 1 mM SA or mock (H2O) at subjective dawn (LL24) or dusk (LL36). The seedlings were harvested and frozen in liquid nitrogen 6 h after treatment.
RNA extraction and cDNA synthesis
Seedlings were manually ground to a fine powder in liquid nitrogen, before being homogenized in RNA extraction buffer (100 mM LiCl, 100 mM Tris pH 8, 10 mM EDTA, 1% SDS). An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added, followed by votexing and centrifuging at 13,000 rpm for 5 min. The aqueous phase was transferred to a tube containing an equal volume of chloroform/isoamyl alcohol (24:1), followed by vortexing and centrifuging at 13,000 rpm for 5 min. This step was repeated before the aqueous phase was transferred to a tube containing 1/3 vol of 8 M LiCl and incubated overnight at 4 °C. The samples were centrifuged at 13,000 rpm at 4 °C for 15 min. The supernatant was removed and the pellet was washed twice in ice-cold (−20 °C) 70% ethanol. The pellet was then dissolved in 400 µl H2O for 30 min on ice. 40 µl of NaAc (pH 5.3) and 1 ml of ice-cold 96% ethanol was added and the solution was incubated for at least 1 h at −20 °C. The tubes were centrifuged at 13,000 rpm for 15 min at 4 °C and the pellet was washed twice with ice-cold 70% ethanol. The final pellet was dissolved in 50 µl H2O. The resulting RNA was quantified using a NanoDrop spectrophotometer and diluted to standardize the concentrations across all samples. SuperScript II reverse transcriptase (Invitrogen) was used to perform reverse transcription according to the manufacturer’s instructions.
Quantitative PCR
For SA-induced transcript levels in wild-type over 48 h (Fig. 4 and Supplementary Fig. 4), cDNA was diluted 20-fold and qPCR was performed in a 5 µl reaction using SYBR Green and gene specific primers on a QuantStudio 5 PCR machine.
For SA-induced transcript levels in clock mutants at subjective dawn and dusk (Fig. 5), cDNA was diluted 20-fold and qPCR was performed in a 10 µl reaction using SYBR Green and gene specific primers on a StepOne Plus Real Time PCR machine.
Circadian rhythmicity analysis of transcript levels
SA-induced PR1 transcript levels in wild type over 48 h (Fig. 4 and Supplementary Fig. 4) were assessed for circadian rhythmicity using the JTK_cycle algorithm with empirical calculation of p-values (eJTK) in BioDare235,50. The mean transcript levels of the two biological replicates were used for analysis with parameters set as eJTK Classic, linear detrending of the input data and p < 0.05.
Pseudomonas disease assay
Col-0, CCA1ox, cca1lhy, TOC1ox and toc1-101 were grown on soil under LD conditions (see plant materials and methods). At 3.5 weeks old, plants were released into LL. At subjective dawn (LL24), plants were sprayed with 1 mM SA or mock (water). At LL48, 2 leaves (leaves 4-6) were pressure infiltrated with Pseudomonas syringae pv. maculicola (Psm) ES4326 at OD600 = 0.005, using a 1 ml syringe. The bacteria were left to infect the plants for 3 days, before leaf disks were harvested from the infiltrated leaves. The leaf disks were homogenized and diluted in 10 mM MgSO4, then plated onto LB plates (10 mM MgSO4 and 50 µg/ml streptomycin). Plates were incubated for 2 days at 28 °C, before calculating colony forming units (CFU) per leaf disk.
Responses