CIRCADIAN CLOCK-ASSOCIATED 1 regulates ROShomeostasis and oxidative stress responsesAlvina Grace Laia, Colleen J. Dohertyb, Bernd Mueller-Roeberc,d, Steve A. Kayb, Jos H. M. Schippersc,d,1,and Paul P. Dijkwela,1

aInstitute of Molecular BioSciences, Massey University, Palmerston North 4442, New Zealand; bSection of Cell and Developmental Biology, Division ofBiological Sciences, University of California at San Diego, La Jolla, CA 92093-0130; cDepartment of Molecular Biology, Institute of Biochemistry and Biology,University of Potsdam, 14476 Potsdam-Golm, Germany; and dMax Planck Institute of Molecular Plant Physiology, 14476 Potsdam-Golm, Germany

Edited by Joseph S. Takahashi, The Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, and approvedSeptember 10, 2012 (received for review May 30, 2012)

Organisms have evolved endogenous biological clocks as internaltimekeepers to coordinate metabolic processes with the externalenvironment. Here, we seek to understand the mechanism ofsynchrony between the oscillator and products of metabolismknown as Reactive Oxygen Species (ROS) in Arabidopsis thaliana.ROS-responsive genes exhibit a time-of-day–specific phase of ex-pression under diurnal and circadian conditions, implying a role ofthe circadian clock in transcriptional regulation of these genes. Hy-drogen peroxide production and scavenging also display time-of-dayphases.Mutations in thecore-clock regulator,CIRCADIANCLOCKASSOCIATED 1 (CCA1), affect the transcriptional regulation of ROS-responsive genes, ROS homeostasis, and tolerance to oxidativestress. Mis-expression of EARLY FLOWERING 3, LUX ARRHYTHMO,and TIMING OF CAB EXPRESSION 1 affect ROS production and tran-scription, indicating a global effect of the clock on the ROS network.WeproposeCCA1as amaster regulator of ROShomeostasis throughassociation with the Evening Element in promoters of ROS genes invivo to coordinate time-dependent responses tooxidative stress.Wealso find that ROS functions as an input signal that affects the tran-scriptional output of the clock, revealing an important link betweenROS signaling and circadian output. Temporal coordination of ROSsignaling by CCA1 and the reciprocal control of circadian output byROS reveal a mechanistic link that allows plants to master oxidativestress responses.

redox homeostasis | transcriptional coordination

Circadian rhythms are directed by day–night cycles so thatorganisms can synchronize external conditions with internal

metabolism to allow temporal separation of incompatiblemetabolicevents (1). Plants undergo aerobic metabolism, e.g., photosynthesisand respiration, which results in the generation of toxic by-productsof oxygen (O2) known as Reactive Oxygen Species (ROS) (2). Thephotoreduction of O2 to H2O gives rise to singlet oxygen, super-oxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radical(·OH) (3). If the production of ROS is left unmanaged, plants mayexperience oxidative stress due to an imbalance in cellular redoxstate that eventually leads to cell death (2). Thus, plants haveevolved enzymatic and nonenzymatic scavenging machineries tokeep ROS at physiologically permissive levels (4).The activation and monitoring of stress-responsive pathways are

energetically demanding. Not surprisingly, circadian gating of stresspathways has been found to confermaximal tolerance to stress whileminimizing the use of resources (5, 6). The Arabidopsis circadianclock consists of a core feedback loop that connects morning- andevening-phase circuits (7). The core loop ismadeupof twomorning-expressed Myb transcription factors (TFs)—CIRCADIAN CLOCKASSOCIATED1 (CCA1) andLATEELONGATEDHYPOCOTYL(LHY)—that inhibit the expression of an evening-expressed pseu-doresponse regulator TIMING OF CAB EXPRESSION 1 (TOC1)through the association of CCA1 to the Evening Element (EE)motif in the TOC1 promoter (8, 9). The morning loop consists ofTOC1 homologs, the PSEUDO-RESPONSE REGULATOR (PRR)

7 and PRR9, which are partially redundant in repressing the tran-scription of CCA1 and LHY (10). In the evening loop, EARLYFLOWERING (ELF) 3, ELF4, and LUX ARRHYTHMO (LUX)driveCCA1 andLHY expression and are required for clock functionunder constant light (LL) (11–13).Because ROS can be used as important secondary messengers

during stress (3, 14), itmay be advantageous forROShomeostasis tobe in tune with daily light–dark cycles to enhance fitness. Moreover,as photosynthesis is driven by the sun, ROS levels would fluctuateacross the day, and organisms may evolve systems to cope with pe-riodic increase in such toxic products. However, the underlyingmechanisms and the biological importance of restricting stressresponses to certain times of the day have not been fully elucidated.Here, we show that the circadian clock coordinates ROS homeo-stasis and transcriptional response. The mechanisms for signalingcircadian time to a proposed clock-controlled output, the ROSpathway, are discussed.

ResultsROS Production and Scavenging Are Regulated by Diurnal Cycles.Photosynthetic genes peak at Zeitgeber time (ZT) 4 (15), sug-gesting that, at this time, light-harvesting capacity peaks, and suchmetabolic changes may tilt the balance of ROS production andscavenging. Thus, we hypothesized that ROS production andscavenging may exhibit time-of-day–specific changes. To test this,H2O2 levels were quantified from plants grown under 12-h light/12-h dark (LD) photocycles. Indeed, H2O2 production peaks atnoon, ZT7 [0.117± 0.009 μmol/mg freshweight (FW)] and reachestrough levels (0.015± 0.008 μmol/mg FW) at midnight, ZT19 (Fig.1 A and C). We also determined the time-of-day activity of theH2O2 scavenger catalase in LD-grown plants. As expected, cata-lase activity peaks at ZT7 (0.611 ± 0.021 U/mL) and dips at ZT19(0.156 ± 0.039 U/mL; Fig. 1B), coinciding with the peak andtrough of H2O2.As we observed diurnal rhythms in ROS production and scav-

enging, we tested whether genes from the ROS network (16) areregulated similarly.Weobtained time-course expression profiles of167 ROS-responsive genes (Dataset S1), selected from a group ofgeneral oxidative stress response markers (16), by quantitativePCR (qPCR). Of the 167 genes, 140 genes display time-of-day–specific phases (one-way ANOVA, P < 0.0001) under long-dayphotocycles (Fig. 1D). As expected, the largest gene cluster peaks

Author contributions: A.G.L., B.M.-R., J.H.M.S., and P.P.D. designed research; A.G.L., C.J.D.,and J.H.M.S. performed research; S.A.K. contributed new reagents/analytic tools; A.G.L.,C.J.D., J.H.M.S., and P.P.D. analyzed data; and A.G.L., C.J.D., B.M.-R., S.A.K., J.H.M.S., andP.P.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. E-mail: p.dijkwel@massey.ac.nz or schippers@mpimp-golm.mpg.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1209148109/-/DCSupplemental.

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at noon (Fig. 2E), coinciding with the peak of ROS levels (Fig. 1 Aand C). To ascertain other biological processes that might becoregulated by these genes, we analyzed Gene Ontology (GO)enrichment and found that genes associated with stresses andabiotic stimuli are overrepresented (Dataset S2).

Circadian Clock Regulates ROS Production, Scavenging, andTranscription of ROS-Responsive Genes. The phase relationship be-tween ROS production, scavenging, and ROS-driven transcrip-tion suggests coordinated regulation of this network within thediel cycle. We next determined whether the observed effectpersists in LL conditions, indicating clock regulation. It is possiblethat H2O2 levels remain high in LL due to the overreduction ofelectron acceptors (17). Indeed, H2O2 levels are elevated in bothsubjective day and night in LL (Fig. 2 A and C). Interestingly, westill observed a time-of-day–specific peak at midday (ZT7; one-way ANOVA, P < 0.001) albeit with a lower peak:trough ratio of1.49 (Fig. 2A) compared with the peak:trough ratio in LD of 7.86.Likewise, catalase activity, although elevated in LL (Fig. 2B), stillshows a significant time-of-day peak with a reduced peak:troughratio in LL (1.59) compared with LD (3.92). To test if the cir-cadian clock exerts transcriptional coordination over ROS sig-naling, expression profiling of the same 167 genes was performedon LD entrained plants that were transferred to LL. We observedthat ROS genes are phased to the subjective midday (ZT10; one-way ANOVA, P < 0.0001) under LL (Fig. 2D), and this impliesa clock-regulated effect. Under LD condition, only 39% of thecycling ROS genes exhibit a noon phase (ZT10) whereas in LL,over 75% of cycling genes peak at the subjective midday (Fig.

2E). Taken together, these persistent time-of-day signals in LL(Fig. 2D) suggest that the circadian clock regulates the ROStranscriptional response network.

Functional Clock Is Required for the Time-of-Day–Specific Regulationof ROS Production. To further investigate the role of the clock inROS signaling, we examined the sensitivity of clock mutants toROS-generating agents. Plants with mutations in CCA1, LHY,ELF3, ELF4, LUX, TIME FOR COFFEE (TIC) (18), PRR5,PRR9, and PRR7 are hypersensitive (Fig. 3 and Fig. S1) to theapplication of 5 μM methyl viologen (MV), which causes an in-crease in superoxide levels (19). In contrast, plants overexpressingCCA1 (CCA1-ox) (8) are hyposensitive to MV (Fig. 3). The ob-served hypersensitivity of cca1-1, lhy-11, and cca1-1/lhy-11mutantsto ROS-generating agents could be a result of impaired ROS ho-meostasis in these genotypes. Under both LD and LL conditions,cca1-1/lhy-11 mutants exhibit higher H2O2 and lower catalaselevels compared with WT plants (Fig. 4 A–C). Interestingly,overexpression of CCA1 suppresses H2O2 levels under both LDand LL (Fig. 4 A and C). However, catalase activity in CCA1-oxplants is found to be lower than WT levels only during the day (inLD) and the subjective day (in LL; Fig. 4B). Both cca1-1 and lhy-11single mutants exhibit high H2O2 levels in the evening and night inLD but not in LL (Fig. S2 A and C). Catalase activity is also de-creased in cca1-1 and lhy-11 single mutants during the day in LDand the subjective day in LL (Fig. S2B). As previously described(20), all three catalase genes—CAT1, CAT2, and CAT3—display

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Fig. 1. ROS homeostasis is regulated by diurnal cycles. (A) H2O2 levels and(B) catalase enzyme activity from 16-d-old Col-0 plants entrained in LD. Errorbars represent SEM of n = 20. One-way ANOVA (effect of time) for LDprofiles of H2O2 and catalase was significant (****P < 0.0001). (C) Images ofH2O2 accumulation in plants stained with 3,3-diaminobenzidine. (D) Ex-pression profiles of 167 ROS transcripts from 16-d-old Col-0 plants (n = 15)entrained in LD. One-way ANOVA (effect of time) for gene expression wassignificant (***P < 0.0001). White bars: day; black bars: night.

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Fig. 2. The circadian clock regulates ROS homeostasis. (A) H2O2 levels and(B) catalase enzyme activity quantified from 16-d-old Col-0 plants entrainedin LD and transferred to LL before sample collection. Error bars representSEM of n = 20. One-way ANOVA (effect of time) for H2O2 and catalase wassignificant (****P < 0.0001, ***P < 0.001). (C) Images of H2O2 accumulationin plants stained with 3,3-diaminobenzidine. (D) Expression profiles of 167ROS transcripts from 16-d-old Col-0 plants (n = 15) entrained for in LD andtransferred to LL before sample collection. One-way ANOVA (effect of time)for gene expression was significant (***P < 0.0001). (E) Genes exhibitingtime-of-day–specific phases were separated into phase clusters. White bars:day; hatched bars: subjective night.

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time-of-day–specific phases in LL. CAT1 and CAT3 peak at noon(ZT7) whereas CAT2 peaks at dawn (ZT3; Fig. 4D). Similar toprevious findings, we observed that eitherCCA1 overexpression ormutations in CCA1 and LHY result in altered CAT1, CAT2, andCAT3 expressions (Fig. 4D) (8, 21). Loss of function in clock genesmay cause circadian arrhythmia (22), and, to investigate globalclock effects on ROS production, H2O2 levels were determined inmutants that have defective clocks. Indeed, H2O2 levels are high inelf3-1, elf4-101, and tic-1 (Fig. S2C). Temporal expression profilingrevealed that CAT1, CAT2, and CAT3 expression is arrhythmic inelf3-1 (Fig S2D) and that the expression ofCAT genes is changed inboth lux-1 and toc1-1 (Fig. S2C). Thus, our results are consistentwith the suggestion that the differential sensitivity of clockmutantsto oxidative stress reflects perturbations in the basal cellular ROShomeostasis in these mutants, although the relationship with MVsensitivity and circadian effects appears complex.

CCA1 Regulates the Coordinated Transcription of ROS Genes in theAbsence of Oxidative Stress. To determine whether the circadianclock regulates the ROS network at the transcriptional level, weinvestigated whether genes grouped under various ROS-relatedGO categories (referred to as ROS GO genes hereafter) in Ara-bidopsis exhibit circadian rhythmicity.We observed that, of the 517ROS GO genes, on average 73 and 39% of the genes are rhythmic

in two or more diurnal and circadian conditions, respectively(Dataset S3). Of these rhythmic genes, the midday phase (ZT5) isfound to be overrepresented in ROS GO genes (Z-score > 1.96;Fig. S3A) when cycling calls were made on two LD datasets(LDHH_SM and LDHH_ST). Furthermore, cycling calls on twoshort-day (8 h light/16 h dark) datasets (COL_SD and LER_SD)revealed that the ZT4 phase is enriched (Z-score> 1.96; Fig. S3B).These results are consistent with an independent study where 34%of ROS genes were found to be clock-regulated (5).To investigate whether this time-of-day expression of ROS genes

is regulated by central components of the circadian clock, we ex-amined the expression of a subset of ROS genes in circadian clockmutants. We performed a 48-h expression profiling on 32 selectedtranscripts in LD entrained plants released to LL in elf3-1, lux-1,toc1-1, cca1-11/lhy-11, and CCA1-ox. These 32 ROS genes are se-lected on the basis of their involvement in ROS signaling (DatasetS4). Of the 32 genes, 24 genes exhibit time-of-day–specific phases inWT plants (one-way ANOVA, P < 0.001; Figs. S4A and S5A;Dataset S5). No overt phases could be detected in the remainingeight genes (Fig. S6). These 24 genes can be grouped into threeclusters based on the time when they peak, i.e., midday (ZT7),evening (ZT11), and predawn (ZT23; Figs. S4A and S5A). Genes ofthe phenylpropanoid pathway are known to peak at predawn (15).Indeed, we observed that the ascorbate biosynthesis geneVITAMINC 2 (VTC2) (3) has a predawn phase (Fig. S4B). Phase-specific ex-pression ofVTC2 is altered inCCA1-ox and cca1-1/lhy-11 (Fig. S4B).Another ROS scavenger is ascorbate peroxidase (APX), and de-ficiency in this enzyme results in light-induced necrosis (23). Thearrhythmic expression profiles of APX4 in CCA1-ox and cca1-1/lhy-11 are distinct from the expression profile in WT plants wherea midday phase (ZT7) can be observed (Fig. S4B). The phase ofAPX4 appears to be shifted in elf3-1 and lux-1 (Fig. S5B).Otherwell-studied genes that exhibit phase-specific expression are the predawnphased (ZT23) HEAT SHOCK PROTEIN 18.2 (HSP18.2), themidday phased (ZT7) PHENYLALANINE AMMONIA-LYASE 1(PAL1), AT2G22420 (PEROXIDASE) and HEAT SHOCKTRANSCRIPTION FACTOR A4A (HSFA4A), and the evening-phased (ZT11) MYB DOMAIN PROTEIN 59 (MYB59; Fig. S4B)(24–27). All five genes show dramatic changes in time-of-day ex-pression inCCA1-ox, cca-1/lhy-11, and elf3-1 (Fig. S4B and S5B). Inall elf3-1, lux-1, and toc-1, midday-phased genes show altered ex-pression (Fig. S5A). Genes from the evening and predawn cluster

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Fig. 3. Mutations in CCA1 and LHY resulted in ROS hypersensitivity. Plantswere entrained in LD and transferred to LL for a day before 5 μM MVtreatment was administered at ZT3 day 16. (A) Mean number of wilted leaveswas scored after 24 h of treatment. Error bars represent SEM of n = 15.Student’s t tests were significant (***P < 0.0001 and **P < 0.05). (B) Pheno-types of treated WT and mutant plants after 24 h.

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Fig. 4. H2O2 and catalase rhythms are regulated by CCA1. (A) H2O2 levels and (B) catalase enzyme activity from 16-d-old WT, CCA1-ox, and cca1-1/lhy-11plants entrained in LD (Left) and transferred to LL (Right). Error bars represent SEM of n = 20. One-way ANOVA (effect of group; mutant versus WT) for H2O2

and catalase was significant (***P < 0.0001, **P < 0.01). (C) Images of H2O2 accumulation in CCA1-ox and cca1-1/lhy-11 plants stained with 3,3-dia-minobenzidine. (D) Expression profiles of CAT1, CAT2, and CAT3 from 16-d-old Col-0, Ler-0, CCA1-ox, and cca1-1/lhy-11 plants (n = 15) entrained in LD andtransferred to LL before sampling. One-way ANOVA (effect of time in Col-0 and Ler-0) for CAT1, CAT2, and CAT3 expressions was significant (***P < 0.001).White bars: day; black bars: night, hatched bars: subjective night.

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appear to display altered amplitudes in toc1-1 (Fig. S5A). In elf3-1,evening and predawn genes are arrhythmic (Fig. S5A). Evening-phased genes show altered phase of expression in lux-1, whereas thepredawn cluster still appears to show a broadened peak (Fig. S5A).In general, phase-specific expressions of the remaining genes fromall three clusters are altered in CCA1-ox and cca1-1/lhy-11 (Fig.S4A), suggesting that rhythmic oscillation in CCA1 levels may beessential for transcriptional coordination of ROS genes.Seven of the eight ROS genes did not display any specific time-

of-day phase in the conditions tested. Nevertheless, they exhibitaltered expression in CCA1-ox, cca1-1/lhy-11, elf3-1, lux-1, andtoc1-1 plants (Fig. S6). This may be the cause of altered ROShomeostasis in these mutants (Figs. 3 and 4 A–C; Figs. S1 andS2C). This also suggests a potential for regulation of the ROSnetwork that is beyond circadian regulation of rhythmic ROSgenes. PYRIDOXINE BIOSYNTHESIS 1 (PDX1) encodes anenzyme involved in production of the ROS quencher pyridoxinethat increases in levels upon UV-B irradiation (28). Notably, theexpression of PDX1 is elevated in cca1-1/lhy-11 (Fig. S6). It isalso interesting that the expression of RESPIRATORY BURSTOXIDASE HOMOLOG C (RBOHC) (29) is elevated in CCA1-oxand toc1-1mutants (Fig. S6). Collectively, the results suggest thatthe circadian clock mediates the expression of ROS-signalinggenes under regular growth conditions.

CCA1 Regulates Responses to Oxidative Stress. The circadian clock inplantsmay regulate phase-specific expression ofROSgenes to allowthe anticipation of oxidative stress according to a diurnal schedule.Of the 32 genes tested in the previous section, 28 had a putative EEand/or CCA1-binding site (CBS) in their upstream promoterregions (Dataset S5). This, combined with the altered expression inCCA1-ox and cca1-1/lhy-11 mutants, prompted us to investigate ifthese responses weremediated directly byCCA1. IfCCA1mediatesthis response, it would depend on the time ofCCA1 expression, i.e.,at dawn. We focused on seven genes (Dataset S5), five of which areROS TFs that rapidly respond to ROS treatments (30, 31), and firstexamined their expression in LL. Time-of-day specific phasing isobserved in six of the seven genes, and these genes also display al-tered expression in CCA1-ox and cca1-1/lhy-11 plants (Fig. 5A). To

determine if CCA1 mediates the response to oxidative stress, weinduced oxidative stress in WT plants, using 2 μM MV, at threedifferent times in a single LD cycle, i.e., morning (ZT3), evening(ZT11), and midnight (ZT19). For all genes tested, treatments inthemorning, but not eveningornight, result in significant inductions(Student’s t test, P < 0.0001; Fig. 5B), consistent with the predictionthat the system is themost responsive in the morning whenCCA1 isexpressed. Interestingly, for six genes, down-regulation (Student’s ttest, P < 0.01) of gene expression is observed in evening-treatedplants (Fig. 5B). In the CCA1-ox background, five of the genes thatare repressed at ZT11 in WT plants show significant inductions inCCA1-ox (Fig. 5C). Furthermore, the diurnal response to ROS,observed in WT plants with the peak response at ZT3 (Fig. 5B), isabolished in CCA1-ox (Fig. 5C). The response to different con-centrations of MV is also attenuated in CCA1-ox (Fig. 5C and Fig.S7).Moreover, we noted that the expression of all seven genes is, inpart, also affected by mutations in ELF3, LUX, and TOC1 underdiurnal conditions (Fig. S8). These mutants have altered circadianclocks; thus, the observed changes in gene expression support theimportance of circadian regulation of these genes. In short, theobserved diurnal response of ROS TFs suggests that plants may bemost responsive to ROS treatments in the morning when CCA1 isexpressed (Fig. 5B).

WRKY11, MYB59, PAL1, and ZAT12 Are Direct Targets of CCA1 in Vivo.We next determined whether CCA1 could physically associate withthe promoters of ROS genes in vivo.WRKY11,MYB59, PAL1, andZAT12 have a time-of day–specific expression that is altered inplants with mis-regulated CCA1 (Fig. S4B and Fig. 5A). This ob-servation and the presence of EE and/or CBS in their promoterregions (Fig. S9 A and C) suggest that CCA1 may be a direct reg-ulator of these genes. We performed chromatin immunoprecipita-tion (ChIP)–qPCR assays using pCCA1::CCA1-GFP transgeniclines at the peak of CCA1 expression (ZT1) with CAT3 (32) asa positive control (Dataset S5). In addition to the four aforemen-tioned genes, six additional ROS genes involved in transcriptionalregulationwere assayed, and enrichments are detected forEE/CBS-containing promoter fragments of COR27 (33), JUMONJI DO-MAIN PROTEIN 5 (JMJD5) (34), ZAT12, MYB59, WRKY11, and

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Fig. 5. Response to ROS is regulated by diurnal cycles and is dependent on the time of CCA1 expression. (A) Expression profiles of seven ROS TFs obtainedfrom plants (n = 15) entrained in LD and transferred to LL before sampling. One-way ANOVA (effect of time) was significant in WT samples (**P < 0.001).Normalized expressions of ROS TFs in (B) Col-0 and (C) CCA1-ox plants, entrained in LD and treated with 2 μMMV at ZT3, ZT11, and ZT19. Error bars representSEM of n = 15. In Col-0 samples (B), Student’s t tests for the effects of time were significant (**P < 0.0001; *P < 0.01). In CCA1-ox samples (C), Student’s t testsfor effects of genotypes (Col-0 versus CCA1-ox) were significant (**P < 0.0001; *P < 0.01). White bars: day; hatched bars: subjective night.

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PAL1 and not in negative control sequences (Fig. S9B). However,enrichment is not observed inPUP1,PEROXIDASE, andMETHYLESTERASE 18 (MES18) although all three genes contain putativeCBS sequences (Fig. S9D).

ROS Signals Feed-Back to Affect a Clock Output.Because interlockingfeedback loops are common in other metabolic processes (35), wehypothesized that ROS signals may feed-back to affect processescontrolled by the oscillator. Such feedback may allow the plant toreset the ROS-signaling cascade through crosstalk with otherpathways, and plants could continuously monitor the changes inROS levels under various physiological conditions. To investigatethe effects of ROS on circadian output, CHLOROPHYLL A/B-BINDING PROTEIN::LUCIFERASE (CAB2::LUC) and FLAVINBINDING, KELCH REPEAT, F-BOX1 (FKF1::LUC) were used.These output genes were selected on the basis of both being likelytargets of CCA1 and containing the EE in their promoters (8, 36–38). ROS treatments were achieved by administering differentdoses of MV, H2O2, the catalase inhibitor 3-aminotriazole (39),and the peroxidase inhibitor salicylhydroxamic acid (40). As ROSsignaling is mediated by both production and scavenging (14), in-hibition of ROS production can be expected to have similar effectsto inducing ROS production. Diphenyleneiodonium chloride andpotassium iodide (41) were used to inhibit NADPH oxidase ac-tivity and to scavenge H2O2, respectively. We observed that FKF1shows a dramatic response to chronic ROS treatments. The FKF1reporter shows phase delays and dose-dependent lengthening ofthe FKF1 period (Fig. S10A). The period and phase of CAB2 (Fig.S10B) are, however, not significantly affected by either ROS in-duction or inhibition. Our results suggest that the clock is involvedin coordinated transcriptional regulation ofROS genes with CCA1being a likely master regulator of ROS networks. In addition, weobserved that ROS could feed-back to affect the transcription ofa clock-regulated output, FKF1.

DiscussionHere we examine the potential crosstalk between the circadian clockand the ROS transcriptional network. Our results provide evidencethat ROS production, response, and transcriptional regulation ofROS genes are controlled by the circadian clock. ROS genes showtime-of-day–specific expression patterns that persist in constantconditions and that this temporal regulation of ROS is also reflectedin the enzymatic activity of catalase and the production of H2O2(Figs. 1 and 2). The changes in phenotypic responses to oxidativestress in plants containing mutations in the components of the cir-cadian clock reflect the importance of the circadian clock in regu-lating this response (Fig. 3 and Fig. S1). Further investigation revealsthat the loss of CCA1 and LHY impairs time-of-day–specific ROSproduction and scavenging (Figs. 4 and 5B). Moreover, the expres-sion pattern of ROS genes is coordinated in a diurnal and circadianmanner that is dependent on components of the circadian clock(Figs. S4 and S5). This coordinated regulation, combined with thehypothesis that ROS levels may reflect the metabolic state of theplant (42), suggests that metabolic needs may be partitioned to dif-ferent times of the diel cycle.Notably, transcriptional coordination ofROS genes may be driven in part by CCA1 rhythms per se (Fig. S4).Although the sensitivity to MV and the ROS production levels

in circadian mutants were altered compared with WT plants,a direct correlation between period effects in the mutants andROS responses was not found. The complex feedback betweenthe circadian components themselves could contribute to suchlack of linearity. For example, in a short-period mutant, CCA1will be expressed at different times of the day than expected, andsensitivity of plants depends on the time-of-day of CCA1 expres-sion (Fig. 5B). Combined with this, the pleiotropic phenotypesof some of the circadian mutants (elf3-1 and lux-1) could causethe connection to be skewed, making it complicated to untanglethe relationship between ROS response and the circadian clock.

Indeed, the lack of a linear relationship between ROS sensitivityand circadian period is reflected by the differential effects of ROStreatments on the circadian output genes CAB2 and FKF1. Thissuggests a complicated role potentially involving multiple oscil-lators and tissue specificity. If this is the case, endogenous ROSproduction and scavenging may become out of sync in differentways in the various circadian mutants.A metaphorical “gate” governs the sensitivity of the clock to

resetting signals presented at different times of the day (43).CCA1regulates the expression of ROS genes only when it is expressed atdawn, and, if oxidative stress is administered at night when CCA1levels are low, this effect is not observed (Fig. 5B). However, theresponse is not limited to dawn in CCA1-ox plants, indicating thatCCA1 is an important component of this gate. Overexpression ofCCA1 appears to enable a continually permissive state for ROSresponses (Fig. 5C) and could therefore account for the low basalROS levels in this mutant (Fig. 4A andC). These lower basal ROSlevels or the overexpression of CCA1 could cause the attenuatedinduction of ROS genes as observed in CCA1-ox in response tooxidative stress (Fig. 5C and Fig. S7). Further analysis will benecessary to determine the causative signaling pathway regulatingthese transcriptional responses.The circadian clock may receive inputs from multiple metabolic

pathways to use this information to fine-tune clock function (44,45). Thus, it is possible for ROS signals to exert indirect effects onclock output pathway(s) when the oscillator is perturbed. Notably,transcription of FKF1 is altered by ROS treatments (Fig. S10A).Both FKF1 and CAB2 were selected on the basis of both beingCCA1-regulated outputs. The identification of the effects of ROSon the expression of FKF1, a known regulator of flowering time(46), could also provide a potential link between previouslyreported studies on antioxidants and flowering. For example, thevtc1mutant has alterations in flowering time that are photoperiod-dependent (47), perhaps due to the accumulation of ROS and itseffect on FKF1 expression. Also, when the antioxidant ascorbicacid was increased artificially, flowering could be delayed, and thiscorrelates with lower mRNA levels of circadian clock and photo-periodic genes (47). FKF1 is also involved in the degradation ofTOC1 through the interactionwithGIGANTEA (GI). Indeed, therole of GI in oxidative stress tolerance has been implicated (48).PRR5, PRR7, and PRR9 have been linked to the regulation of ROSproduction (49) where the prr5-1, prr9-1, prr7-3, and prr5-1/prr9-1mutants are hypersensitive to ROS treatments (Fig. S1). In-terestingly, these mutants also have altered levels of CCA1 (50),which possibly explains the mechanism of this effect.The observed effect of ROS on the expression FKF1 but not on

CAB2 suggests that the effects of ROS-generating agents on thecircadian output are not uniform. This disconnection could resultfrom the presence of multiple oscillators or tissue-specific differ-ences in the expression of FKF1 and CAB2 (51, 52). FKF1 isexpressed in both vascular bundles and mesophyll cells whereasCAB2 is expressed in mesophyll cells and epidermal guard cells(53, 54). It was previously observed that CAB2 and CAT3 areregulated by two oscillators within the same tissue (51). Both genesrespond differently to temperature signals although the spatialexpression patterns of CAB2 and CAT3 overlap in the mesophyll.The toc1-1 mutant has short period rhythms of CAB2 but hasa wild-type period for cytosolic Ca2+ oscillation whereas the toc1-2mutant has a short period for bothCAB2 and Ca2+ rhythms, whichsuggests that different mechanisms regulate the rhythms of CAB2andCa2+ (52).We contemplated the possibility that becauseCAB2is involved in photosynthetic responses and may be exposed toROS fluctuations, its rhythms are buffered fromROS responses bysome mechanism—perhaps a second oscillator. It is also note-worthy that CAB2 is repressed by glucose and fructose (55), andtherefore it is possible forCAB2 rhythms to be buffered from otherproducts of photosynthesis. Alternatively, the differential respon-ses between FKF1 and CAB2 reporter constructs could also be

Lai et al. PNAS | October 16, 2012 | vol. 109 | no. 42 | 17133

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due to the time of day at which the genes are expressed. BecauseCCA1 repressesmany targets likeFKF1 that are expressed at night,whereas CCA1 appears to be a positive regulator of CAB2 (8, 37,38), the differences in ROS response could be due to differentmechanisms of CCA1 action on CAB2 versus FKF1.Recently, an enzyme that senses H2O2 has been proposed to

be the ancestor of all biological clocks where it is assumed thatclocks either evolved to confer metabolic advantage to anticipatethe presence of ROS or to cope with periodic fluctuations in UVradiation from the sun (56). Our results have shown the mech-anistic relationship between ROS homeostasis and biologicaltimekeeping, which may have coevolved as a result of the GreatOxidation Event (56). We demonstrate that CCA1 is a centralregulator of the ROS-responsive transcriptional network where

it is essential for the coordinated response to oxidative stress andthe regulation of ROS production and scavenging.

Materials and MethodsDetails are described in SI Materials and Methods. This includes information onplantmaterials, growth conditions, H2O2 and catalase assays, ROShypersensitivityassays, ROS treatments for transcript analysis, ChIP-qPCR assays, bioluminescenceassays, and bioinformatics analyses. Primers used for expression analysis areprovided in Dataset S6.

ACKNOWLEDGMENTS. We thank D. Mertten and J. Jayaraman for technicalassistance and C. R. McClung, D. Hincha, and E. Tobin for gifts of seeds. Wethank the Institute of Molecular Biosciences for funding (A.G.L.) and theBundesministerium für Bildung und Forschung - Golm Forschungseinheit zurSystembiologie Systems Biology Research Initiative for funding (Grant FKZ0313924 to B.M.-R.).

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