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Circadian and Plastid Signaling Pathways Are Integratedto Ensure Correct Expression of the CBF and COR Genesduring Photoperiodic Growth1

Louise Norén, Peter Kindgren2, Paulina Stachula, Mark Rühl, Maria E. Eriksson, Vaughan Hurry, andÅsa Strand*

Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, SE–901 87 Umea, Sweden

ORCID IDs: 0000-0003-0947-2648 (P.K.); 0000-0002-7476-1043 (P.S.); 0000-0002-2661-5535 (M.R.); 0000-0003-2038-4892 (M.E.E.);0000-0001-5151-5184 (V.H.); 0000-0001-6664-0471 (A.S.).

The circadian clock synchronizes a wide range of biological processes with the day/night cycle, and correct circadian regulationis essential for photosynthetic activity and plant growth. We describe here a mechanism where a plastid signal converges withthe circadian clock to fine-tune the regulation of nuclear gene expression in Arabidopsis (Arabidopsis thaliana). Diurnaloscillations of tetrapyrrole levels in the chloroplasts contribute to the regulation of the nucleus-encoded transcription factorsC-REPEAT BINDING FACTORS (CBFs). The plastid signal triggered by tetrapyrrole accumulation inhibits the activity ofcytosolic HEAT SHOCK PROTEIN90 and, as a consequence, the maturation and stability of the clock component ZEITLUPE(ZTL). ZTL negatively regulates the transcription factor LONG HYPOCOTYL5 (HY5) and PSEUDO-RESPONSE REGULATOR5(PRR5). Thus, low levels of ZTL result in a HY5- and PRR5-mediated repression of CBF3 and PRR5-mediated repression of CBF1and CBF2 expression. The plastid signal thereby contributes to the rhythm of CBF expression and the downstream COLDRESPONSIVE expression during day/night cycles. These findings provide insight into how plastid signals converge with,and impact upon, the activity of well-defined clock components involved in circadian regulation.

In a wide range of organisms, the circadian clocksynchronizes biological processes with the time of day.The circadian oscillator provides a robust internalrhythm that anticipates daily changes and optimizes theusage of resourceswith day/night cycles. In Arabidopsis(Arabidopsis thaliana), the clock consists of a repressilatorwith a series of transcription-translation feedback loops(Pokhilko et al., 2012), and up to 70% of the chloroplastand 36% of the nuclear genomes have been shown tobe subject to circadian regulation (Harmer et al., 2000;Schaffer et al., 2001;Michael andMcClung, 2003;Michaelet al., 2008). Correct circadian regulation in plants isimportant for photosynthetic activity and growth bysynchronizing gene expression, proteinmodification, andstomatal opening with the light/dark cycle (Hennesseyand Field, 1991; Green et al., 2002; Dodd et al., 2005). The

mechanisms and signaling pathways that connect thecircadian clock with the regulation of photosynthetic ac-tivity in the chloroplasts are not well known; however,there are some suggested mechanisms by which the cir-cadian oscillator can communicate timing information tothe chloroplast (Dodd et al., 2014). For example, the coresubunits of the plastid-encoded polymerase are encodedby the chloroplast genome, but the nucleus-encodedsigma factors are required for promoter specificity andthe initiation of transcription (Schweer et al., 2010). Thenuclear genome in Arabidopsis encodes six sigma factors(SIG1–SIG6), and their expression is circadian regulated; itwas suggested that the circadian timing of the nucleus-encoded sigma factors in turn controls the timing oftranscription of the photosynthesis genes encoded in thechloroplast (Noordally et al., 2013). This regulatory con-trol of transcription could provide a way to communicatetiming to the chloroplast. Furthermore, it has been sug-gested that the chloroplast itself is involved in the regula-tion of the circadian clock and that chloroplast retrogradesignals can alter circadian rhythms (Hassidim et al.,2007). Thus, the close interaction between the circadianclock and chloroplast retrograde signaling systems couldprovide fine-tuning of photosynthetic gene expressionand photosynthetic activity during the day.

Expression of the nucleus-encoded C-REPEATBINDING FACTOR (CBF) transcription factors, CBF1,CBF2, and CBF3, is circadian regulated with a peak atthe middle of the light period (Bieniawska et al., 2008;Kidokoro et al., 2009; Dong et al., 2011). The CBFpathway is important for cold acclimation and freezing

1 This work was supported by the Swedish Research Foundation(to Å.S. and M.E.E.) and the Carl Tryggers Stiftelse för VetenskapligForskning (to M.E.E. and M.R.).

2 Present address: Copenhagen Plant Science Centre, University ofCopenhagen, Thorvaldsenvej 40, 1871 Frederiksberg, Denmark.

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Åsa Strand ([email protected]).

L.N., P.K., P.S., V.H., M.E.E., and Å.S. designed the experiments;L.N., P.K., M.R., and P.S. carried out the experiments; all authorsinterpreted the results and contributed to writing the article.

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http://orcid.org/0000-0003-0947-2648

http://orcid.org/0000-0002-7476-1043

http://orcid.org/0000-0002-2661-5535

http://orcid.org/0000-0003-2038-4892

http://orcid.org/0000-0001-5151-5184

http://orcid.org/0000-0001-6664-0471

mailto:[email protected]

http://www.plantphysiol.org

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.16.00374


tolerance in Arabidopsis, and it regulates the expressionof more than 100 COLD RESPONSIVE (COR) genes(Fowler and Thomashow, 2002; Park et al., 2015). Duringunstressed photoperiodic conditions, PHYTOCHROMEINTERACTING FACTOR7 (PIF7) was shown to nega-tively regulate CBF1 and CBF2 expression (Kidokoroet al., 2009). PIF7 is under circadian control and binds tothe G-box element in the promoters of CBF1 and CBF2and represses their expression. In contrast, CBF3 ex-pression is not controlled by PIF7. The downstreamCORgenes, such as COR15a, COR47, and COR78, also havebeen shown to oscillate during the day and to peak justafter the CBFs (Dong et al., 2011). COR15a encodes acryoprotective protein that is targeted to the chloroplaststroma, where it decreases the propensity of the plastidmembranes to form hexagonal II phase structures inresponse to low temperatures (Steponkus et al., 1998). Inaddition to low temperature and the circadian clock,COR15a expression has been shown to respond to con-ditions that alter the functional state of the chloroplast(Lin and Thomashow, 1992; Kleine et al., 2007). How-ever, the mechanism by which the chloroplasts com-municate the signal to regulateCOR15a expression in thenucleus has remained unknown.Several plastid signals have been reported to influence

the transcription of nuclear genes encoding plastid-localized proteins in a process termed retrograde sig-naling (Rodermel, 2001; Fernández and Strand, 2008;Barajas-López et al., 2013a). One of the identified retro-grade signals is linked to the tetrapyrrole biosyntheticpathway, and several reports have shown that pertur-bations of the tetrapyrrole pathway result in majorchanges of nuclear gene expression (Strand et al., 2003;Ankele et al., 2007; Pontier et al., 2007; Mochizuki et al.,2008; Moulin et al., 2008; Zhang et al., 2011). The copperresponse defect (crd) mutant has impaired activity of thecyclase complex, which converts magnesium protopor-phyrin IX-methylester (Mg-ProtoIX-ME) and atomicoxygen to 3,8-divinyl protochlorophyllide in the chlo-rophyll branch of the tetrapyrrole pathway.As a result ofthis lesion, the crdmutant overaccumulates Mg-ProtoIX-ME and the upstream intermediate magnesium pro-toporphyrin IX (Mg-ProtoIX) that, in turn, triggers aplastid signal regulating nucleus-encoded genes (Totteyet al., 2003; Bang et al., 2008). In the wild type, Mg-ProtoIX andMg-ProtoIX-MEwere shown to accumulatetransiently when plants were exposed to oxidative stress(Stenbaek et al., 2008; Kindgren et al., 2011; Zhang et al.,2011), and in response to the tetrapyrrole accumula-tion, the ATPase activity of cytosolic HEAT SHOCKPROTEIN90 (HSP90) proteins is inhibited, which leadsto a change in nuclear gene expression (Kindgren et al.,2012). HSP90 is a molecular chaperone that interactswith proteins in their near-native state and is essential formaintaining the activity of signaling proteins (Younget al., 2001) aswell as preventing the protein aggregationof freshly translated chloroplast preproteins (Fellereret al., 2011). HSP90 also has been reported to be requiredfor proper regulation of the circadian clock. A rolefor HSP90 was established in the direct control of

maturation and stabilization of the F-box proteinZEITLUPE (ZTL; Kim et al., 2011). ZTL is anevening-phased clock component responsible forthe proteasome-dependent degradation of TOC1 andPSEUDO-RESPONSE REGULATOR5 (PRR5), twoevening-phased components of the circadian clock(Más et al., 2003; Kiba et al., 2007). PRR5 was shown tobe a repressor of the CBF genes (Nakamichi et al., 2012).HSP90 effectively binds ZTL and prevents aggregationof the ZTL protein, providing a rhythm of ZTL andcontributing to the oscillations of TOC1 and PRR5 (Kimet al., 2011).

Here, we report that CBF expression during photo-periodic conditions is regulated by the interplay be-tween circadian and plastid signaling pathways. Wedemonstrate that the stability of ZTL, controlled byHSP90, is decreased in the crd mutant that over-accumulates tetrapyrroles. ZTL directly interacts withthe transcription factor LONG HYPOCOTYL5 (HY5)and negatively regulates HY5. Low levels of ZTL re-sult in HY5- and PRR5-mediated repression of CBF3and PRR5-mediated repression of CBF1 and CBF2 ex-pression. Thus, the plastid signal regulates the activityof HY5 and PRR5 via HSP90 and ZTL and therebycontributes to the changes in expression levels of CBFand COR during the day/night cycle. These findingsprovide novel insight into how plastid signals convergewith, and impact upon, the activity of well-defined clockcomponents involved in circadian regulation.

RESULTS

The Expression of COR and CBF Is Negatively Correlatedwith the Accumulated Levels of Tetrapyrroles in thecrd Mutant

The expression of COR genes responds to the circadianclock as well as to abiotic stress and to treatments that alterthe functional state of the chloroplast (Lin andThomashow,1992; Kleine et al., 2007; Nakayama et al., 2007; Dong et al.,2011). We examined the transcript levels for two CORgenes, COR15a and COR47, in the wild type and in thetetrapyrrole-overaccumulating crd mutant. In accor-dance with earlier reports, COR15a and COR47 expres-sion increased in the light period with a peak just beforedusk (Fig. 1; Dong et al., 2011). In tobacco (Nicotianatabacum) leaves, itwas shown that tetrapyrrole levels alsofollow a diurnal oscillation pattern (Papenbrock et al.,1999). Also in Arabidopsis, the amounts of Mg-ProtoIXand Mg-ProtoIX-ME increased rapidly at the beginningof the light period, peaking in the middle of the day;thereafter, the levels of porphyrins decreased until dusk(Fig. 2). COR expression in the crd mutant, which over-accumulates Mg-ProtoIX and Mg-ProtoIX-ME, was sig-nificantly lower compared with that in the wild type,with the largest difference between the genotypes at 9 hafter lights-on, Zeitgeber Time (ZT 9) (Fig. 1).

To investigate whether the misregulation of the CORgenes in the crdmutantwas due to amisregulation of theupstream CBF genes, we checked the expression of the

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CBF genes (CBF1 to CBF3) during the light/dark cycle(Fig. 2A; Supplemental Fig. S1). Analysis of CBF1 toCBF3 expression demonstrated that the crd mutant dis-played repressed CBF expression compared with thewild type at ZT 6 (Fig. 2A; Supplemental Fig. S1). Takentogether, these results suggest that the altered COR ex-pression in the crdmutant is most likely not caused by adirect effect on the regulation of COR15a and COR47expression but, rather, is the result of the misregulationof the CBF transcription factors.

The Plastid Signal Converges with the Circadian Controlof CBF Expression

Under normal photoperiodic conditions, CBF3 ex-pression has been shown to be regulated by componentsin the circadian clock, which also results in a rhythmicexpression pattern of the COR genes (Fig. 1; Dong et al.,

2011). Our results from the crd mutant suggest that aplastid signal triggered by the accumulation of tetra-pyrroles during the day converges with the circadiancontrol of CBF expression. Under constant light or in thedark, Mg-ProtoIX and Mg-ProtoIX-ME have been dem-onstrated conclusively to decrease to very low and stablelevels without any rhythm (Papenbrock et al., 1999).Thus, in order to examine the regulation of CBF ex-pression, and to dissect the circadian and diurnalsignaling pathways, plants grown under photoperi-odic conditions were transferred to constant light andCBF3 expression was investigated in the wild type andcrd. The levels of Mg-ProtoIX were undetectable usingour method following exposure to constant light, andMg-ProtoIX-ME reached levels detected in the darkduring the normal light/dark cycle in both the wildtype and the crdmutant (Fig. 2, B and C). Furthermore,as observed previously, no oscillation pattern wasdetected for Mg-ProtoIX-ME following exposure toconstant light (Fig. 2C).

The wild-type plants showed an oscillation of CBF3expression during the day, peaking 6 h into the lightperiod and then declining, correlating with the tetra-pyrrole accumulation that peaked at the same timepoint (Fig. 2). Following exposure to constant light,the amplitude of CBF3 expression was reduced in thewild type (Fig. 2). This is commonly observed fol-lowing shifts to constant light for clock-regulatedgenes and so also for CBF3 and the COR genes(Dong et al., 2011). However, the circadian oscillationof CBF3 expression was maintained in both the wildtype and crd during the subjective day. Interestingly,the observed difference in CBF3 expression betweenthe wild type and crd under diurnal conditions wasdiminished. The levels of Mg-ProtoIX-ME followingexposure to constant light were similar to what wasdetected at ZT 12 under normal diurnal conditions,and at this time point there was no difference in CBF3expression between the wild type and crd. These re-sults suggest that the oscillation of tetrapyrrole levelsduring diurnal conditions contributes to the regula-tion of CBF3 expression.

The CBF cold-response pathway is of great impor-tance during cold acclimation and the development offreezing tolerance. Our results indicate that the plastidsignal triggered by tetrapyrrole accumulation duringthe day contributes to the regulation of CBF expression.In order to examine whether this plastid signal plays arole in response to low temperature, we performed acold-stress experiment with wild-type plants andshowed that, following a shift to 4°C, the amounts ofMg-ProtoIX and Mg-ProtoIX-ME decreased rapidlywithin the first 1.5 h in the cold and thereafter weremaintained at very low but stable levels (SupplementalFig. S2). This result is consistent with earlier reportsdemonstrating that the chlorophyll biosynthetic path-way is strongly inactivated and the enzymes in thepathway are inhibited by low temperatures (Mohantyet al., 2006). In contrast, CBF1 to CBF3 were stronglyinduced within the first 1.5 h following the shift to 4°C

Figure 1. COR expression is repressed in the crdmutant comparedwith thewild-typeColumbia (Col). Plantswere grown in short-day conditions (9/15hlight/dark), and 3-week-old plants were sampled every 3 h starting at dawnand analyzed for COR15a (At2g42540; A) and COR47 (At1g20440; B)expression. The last samples were collected 3 h into the dark period. Geneexpression was normalized to ubiquitin-like protein (UBI; At4g36800) andrelated to the amount present in wild-type Col at ZT 0. Each data pointrepresents the mean 6 SD of at least three biological replicates.

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and thereafter decreased with exposure time, whileCOR15a expression showed a weak induction after1.5 h but increased with time exposed to 4°C. The in-duction of CBF and COR expression by exposure to lowtemperatures is much greater compared with the di-urnal changes in expression observed during warmcontrol conditions (Supplemental Fig. S2; Fowler andThomashow, 2002).

HY5 Binds to the Promoters of CBF1/2/3 and COR15ain Vitro

The promoters of the CBF genes show high sequencesimilarity, and a 65-bp fragment (2113 to247 from thetranslation start) of CBF2 has been shown to be re-sponsible for the circadian and cold regulation oftranscription (Kidokoro et al., 2009). CBF1 and CBF2have G-boxes (CACGTG; Kidokoro et al., 2009) close tothe transcriptional start site in their promoters, whileCBF3 has two Z-boxes (TACGTG). The promoter ofCOR15a includes several C-repeat elements, the bind-ing site of the CBFs, as well as several G-boxes (Bakeret al., 1994). Several transcription factors are able tobind the G-box and its variants, but we focused on thebZIP-type transcription factor HY5 (Chattopadhyayet al., 1998). HY5 responds to different light signals buthas also been shown to respond to plastid signals(Ruckle et al., 2007; Kindgren et al., 2012). To study ifHY5 interacts directly with the promoters of the CBFgenes and COR15a, recombinant HY5 was expressedin Escherichia coli and purified under native conditions(Fig. 3A). Purified HY5 was detected on an SDS-polyacrylamide gel as a single band at approximately22 kD, close to the predicted mass (18.5 kD + His tag).The purified protein had an A260:A280 ratio of 0.6,indicating negligible contamination of nucleic acids.Recombinant HY5 was incubated with 50-bp fluorescein-labeled fragments of each promoter (CBF1,2110 to260from the translation start; CBF2, 2110 to 261; CBF3,2110 to 261; and COR15a, 2176 to 2126). The inter-action between protein and probe was visualized afteran EMSA, where a shifted band indicated a protein +DNA complex (Fig. 3B). Interaction between HY5 andthe different promoter fragments could be seen in allcases, albeit a stronger binding affinity was found be-tween HY5 and CBF1 and CBF3 compared with CBF2and COR15a (Fig. 3B). The interaction was lost whenanother fragment of the CBF3 promoter that does notinclude a G-box or a Z-box element was incubated withHY5 (Fig. 3C). The affinity of HY5 to theCBF1 andCBF3promoters also was examined with the addition ofa nonlabeled competitor (Fig. 3, D and E). The disap-pearance of the shifted HY5-CBF1 and HY5-CBF3complexes following the addition of a nonlabeled CBF3

Figure 2. The diurnal oscillation of tetrapyrroles contributes to thecircadian control of CBF3 expression. Plants were grown in short-dayconditions (9/15 h light/dark), and 3-week-old plants were sampledevery 3 h starting at dawn until 3 h into the dark period. After the darkperiod, the plants were kept in constant light for 27 h, and then anotherset of samples were collected every 3 h starting 3 h after the subjectivedawn. Black bars on the x axis represent the true dark period, while graybars represent the subjective dark period during the free-running period.A, Samples were analyzed for CBF3 (At4g25480) expression normal-ized to ubiquitin-like protein (At4g36800), and each time point was

related to the amount present in wild-type Col at ZT 6. B and C, Mg-ProtoIX (B) Mg-ProtoIX-ME (C) contents. Each data point represents themean 6 SD of at least three biological replicates. FW, Fresh weight.






promoter fragment was greater than that following theaddition of a nonlabeled CBF1 promoter fragment.Thus, the competitor experiments showed that HY5bound the promoter of CBF3 stronger than the pro-moter of CBF1. Thus, the in vitro assay showed that,while recombinant HY5 is able to recognize the pro-moters of CBF1 to CBF3 as well as the promoter ofCOR15a, it has the strongest affinity for CBF3.

The Repression of CBF3 Is Mediated via HY5

In order to determine whether HY5 regulates the CBFgenes in response to the tetrapyrrole-triggered plas-tid signal, 3-week-old plants were fed Mg-ProtoIX(Supplemental Fig. S3A) and checked for CBF1 to CBF3and COR15a expression. To assess if tetrapyrrole feed-ing resulted in oxidative stress, we investigated theexpression levels of the three marker genes for reactiveoxygen species (ROS), FER1,GST5, andMAPK18 (Laloiet al., 2007). Following feeding, there was no change

in expression of the ROS marker genes, indicating thatno oxidative stress was induced by the treatment(Supplemental Fig. S3B). This suggests that the plas-tid signal is linked to the tetrapyrrole levels ratherthan ROS generated by their accumulation. TheMg-ProtoIX-incubated wild-type plants showed a sig-nificant repression of CBF1 to CBF3 and COR15aexpression compared with the control plants (Fig. 4A;Supplemental Fig. S3C). This is similar to what hasbeen reported for PHOTOSYNTHESIS-ASSOCIATEDNUCLEAR GENE (PhANG) expression in earlier re-ports from Arabidopsis (Strand et al., 2003; Kindgrenet al., 2012; Barajas-López et al., 2013b). In addition,CBF expression was repressed to less than 40% of con-trol levels in response to norflurazon treatment in thewild type. This repression was absent in the genomeuncoupled5 mutant that accumulated less tetrapyrrolescompared with the wild type in response to thenorflurazon treatment (Strand et al., 2003). In contrast tothe wild type, the hy5mutant did not show a repressionof CBF3 or COR15a following Mg-ProtoIX feeding.

Figure 3. HY5 binds to COR15a and CBF1/2/3 promoters, with a preference for the CBF3 promoter. A, HY5 protein wasexpressed and purified as a His-tag fusion protein and used in electromobility shift assay (EMSA). B, Fluorescein-labeled probe(500 pM) was incubated with increasing concentrations of HY5 protein as indicated. C, Negative control for HY5 DNA bindingusing the CBF3 promoter sequence excluding the G-box and Z-box cis-elements. D and E, Competitor experiments with unla-beled CBF1 and CBF3 promoter fragments with labeled CBF1 probe (D) or CBF3 probe (E).


Norén et al.






However, the expression levels of CBF1 and CBF2 inhy5 were identical to those in the wild type followingthe feeding (Fig. 4A), implying that HY5 is onlyinvolved in the regulation of CBF3 expression. The re-sult from the tetrapyrrole feeding is consistent with that

from the EMSA, where the strongest affinity of HY5was shown for CBF3 (Fig. 3).

To test the genetic interaction between HY5 and CRD,we generated a hy5 crd double mutant. The hy5 singlemutant showed increased CBF3 expression comparedwith the wild type at ZT 6 (Fig. 4B). CBF1 and CBF2expression was similar to that in the wild type in the hy5mutant, supporting the tetrapyrrole feeding experimentindicating that HY5 is only involved in the repression ofCBF3 (Fig. 4, A and B). The tetrapyrrole levels in the hy5mutant were similar to wild-type levels, excluding anydirect tetrapyrrole effect in themutant (Table I). If HY5 isdownstream of the plastid signal, the hy5 crd doublemutant should release the suppressed CBF3 expressionin the crd mutant. The hy5 crd double mutant showedsignificantly higher CBF3 expression compared with thecrd single mutant (Fig. 4B). However, hy5 is not com-pletely epistatic to crd, suggesting that another compo-nent also is involved in the regulation of CBF3expression. The tetrapyrrole levels in the hy5 crd doublemutant were close to the levels detected for the crdsingle mutant (Table I); hence, the rescue of the phe-notype in the hy5 crd mutant with regard to CBF3 ex-pression cannot be explained by changed tetrapyrrolelevels. We also tested CBF3 expression in the hy5 mu-tant following exposure to constant light at ZT 57 (Fig.4C), when the accumulated levels of tetrapyrroles weresimilar to those found in the dark under normal diurnalconditions (Fig. 2C). Thus, when the oscillation of tet-rapyrrole levels was lost, the hy5 mutant showed CBF3expression similar to the wild type. Taken together,these results suggest that HY5 is responding to elevatedtetrapyrrole levels and represses specifically CBF3 ex-pression during diurnal conditions.

HSP90 Responds to the Accumulated Tetrapyrroles andInhibits HY5

The accumulation of tetrapyrroles inhibits theATPase activity of HSP90, and it has been shown thatHY5 responds to the inactivation of HSP90 (Kindgrenet al., 2012). In order to examine if HSP90 plays a rolein the photoperiodic control of CBF3 and COR15aexpression, we investigated CBF3 and COR15a expres-sion in two independent Col-hsp90-RNAi lines with30% of the wild-type HSP90 expression levels left

Figure 4. HY5 regulates CBF3 expression in response to the plastidsignal. A, Plants were grown in short-day conditions (9/15 h light/dark),and 3-week-old plants were analyzed for CBF1 to CBF3 (At4g25490,At4g25470, and At4g25480) expression following 12 h of feeding with50 mM Mg-ProtoIX. B, CBF3 (At4g25480) expression 6 h into the lightperiod in wild-type Col, crd, hy5, and hy5 crd. CBF3 expression wassignificantly different in crd and hy5 compared with Col and in hy5 crdcompared with crd. C, Plants were kept in constant light for 27 h, andthen samples were collected 9 h after the subjective dawn at ZT 57 andanalyzed for CBF3 (At4g25480) expression. Following constant lighttreatment, no significant difference could be observed between Col,crd, and hy5 gene expression. All gene expression data were normal-ized to ubiquitin-like protein (UBI; At4g36800) and related to theamount present in wild-type Col. Each data point represents themean6SD of at least three biological replicates. Statistical differences werecalculated using Student’s t test: ***, P , 0.001.

Table I. Mg-ProtoIX and Mg-ProtoIX-ME contents in hy5 and hy5 crd

Plants were grown in short-day conditions (9/15 h light/dark, 22˚C/18˚C, and 150 mE m22 s21), and 3-week-old plants were sampled 6 hinto the light period and analyzed for Mg-ProtoIX and Mg-ProtoIX-MEcontents. Each data point represents the mean 6 SD of at least threebiological replicates.

Plant Mg-ProtoIX Mg-ProtoIX-ME

pmol g21 fresh wtCol 109.4 6 21.7 94.0 6 16.1crd 146.9 6 14.4 837.2 6 94.5hy5 102.0 6 12.5 65.5 6 13.2hy5 crd 188.0 6 20.1 1,168.2 6 71.7





(Kindgren et al., 2012; Fig. 5A). CBF3 expression inthe Col-hsp90-RNAi lines was significantly lower in theRNA interference (RNAi) lines compared with thewild type, supporting the described connection be-tween the plastid signal and HSP90 (Fig. 5A). We alsoincubated crd and hy5 with the specific inhibitor ofHSP90, geldanamycin (GDA). GDA binds specificallyto the ATP-binding site of HSP90 (Whitesell et al.,1994) and has been shown to clearly inhibit the ATPaseactivity of HSP90 (Avila et al., 2006). Wild-type seed-lings showed a clear repression of CBF3 expressionfollowing GDA treatment, confirming that HSP90 ac-tivity is involved in the regulation of CBF3 expression(Fig. 5B). Similar to the wild type, crd also showeda repression of CBF3 expression following GDA

treatment compared with control conditions (Fig. 5B).In contrast, the hy5 mutant was insensitive to the GDAtreatment with regard to CBF3 expression, suggestingthat HY5 acts downstream of HSP90. However, CBF1and CBF2 expression was repressed similar to thewild type in hy5 following GDA treatment, supportingthat HY5 specifically regulates CBF3 expression(Supplemental Fig. S3D).

HY5 Interacts Physically with ZTL in Vivo

HSP90 has been shown to be required for the properfunction of the circadian clock through maturationand stabilization of the clock protein ZTL (Kim et al.,2011). Possibly, ZTL could be the connection betweenHSP90 and HY5 and, thereby, constitute the clockcomponent involved in the photoperiodic control ofCBF3 and COR15a expression. As described previ-ously by Kim et al. (2011) Col-hsp90-RNAi lines withreduced HSP90 levels showed reduced protein levelsof ZTL, which alsowas seen in our lines (SupplementalFig. S5). To test the putative link between the plastidsignal and ZTL, ZTL protein levels were determined inwild-type and crd plants during the light period and3 h after dusk (Fig. 6A). The result showed a significantreduction of ZTL protein levels in the crd mutantcompared with the wild type at all time points investi-gated (Fig. 6B). In addition, the ZTL protein levelsreached the highest amounts in the wild type once thelevels of tetrapyrroles had decreased at ZT 9 and ZT 12(Figs. 2 and 6A). It has been shown previously that ZTLprotein levels oscillate with a peak at the end of the lightperiod and that GIGANTEA is essential to establish andsustain oscillations of ZTL by a direct protein-proteininteraction (Kim et al., 2007). Our data also suggest thatthe oscillations of tetrapyrrole levels and their inactiva-tion of HSP90 contribute to the level of ZTL protein.

To further test the putative link between HY5 andZTL, we determined if there is a direct physicalinteraction between ZTL and HY5. In an in vivocoimmunoprecipitation (Co-IP) assay, ZTL-Myc andHY5-HA fusion proteins were transiently coexpressedin Arabidopsis protoplasts, and Co-IP was performedusing an anti-cMyc monoclonal antibody bound toprotein G-coated magnetic beads. Inputs and Co-IPfractions were detected by immunoblot analysis withanti-c-Myc and anti-HA antibodies, respectively (Fig.6C). HY5 was successfully detected in the ZTL-Mycimmunoprecipitate, whereas only a background bandcould be detected in the control and single transforma-tions. Thus, a direct physical interaction between ZTLand HY5 was observed in Arabidopsis protoplasts.

ZTL, HY5, and PRR5 Regulate CBF3 and COR15aExpression during Photoperiodic Conditions

We investigated CBF3 and COR15a expression inthe ztl-3 mutant, the double mutants ztl crd and ztl hy5,and the ZTL-OX line (Fig. 6D). CBF3 and COR15a

Figure 5. HSP90 responds to the plastid signal and regulates HY5. A,Plants were grown in short-day conditions (9/15 h light/dark), and3-week-old plants were analyzed for CBF3 (At4g25480) expression 6 hinto the light period in Col and two independent hsp90-RNAi lines inthe wild-type background. The expression data were normalized toubiquitin-like protein (UBI; At4g36800) and related to the amountpresent in wild-type Col. CBF3 expression was significantly different inthe Col-hsp90 lines compared with Col. Each data point represents themean 6 SD of at least three biological replicates. B, Plants were grownfor 10 d in short-day conditions and analyzed for CBF3 (At4g25480)expression following 24 h of feeding with 80 mM GDA. The gene ex-pression data were normalized to ubiquitin-like protein (At4g36800)and related to the amount present in the Murashige and Skoog (MS)control for each genotype. Each data point represents the mean6 SE ofat least three biological replicates. Statistical differences were calcu-lated using Student’s t test: *, P, 0.05; **, P, 0.01; and ***, P, 0.001.


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expression in the ztl mutant was very low, which in-dicates that ZTL is required for proper expression un-der warm growth conditions (Figs. 6D and 7A). Incontrast, the ZTL-OX line showed higher CBF3 ex-pression compared with the wild type (Fig. 6D). Thelow expression levels observed in the ztlmutant cannotbe explained by changes in the circadian period; CBF3and COR15a expression was lower compared withwild-type expression in ztl at all time points investi-gated in the light (Supplemental Fig. S6). In addition,the expression of CBF1 and CBF2 also was lower in theztlmutant compared with the wild type (SupplementalFig. S4). Furthermore, the ztl crd double mutant did notshow any additive effect forCBF3 expression comparedwith the ztl single mutant, suggesting that CRD and

ZTL are genetically linked (Fig. 6D). The hy5 ztl doublemutant showed higher CBF3 and COR15a expressioncompared with the ztl single mutant (Figs. 6D and 7A).However, similar to the hy5 crd double mutant, hy5 isnot fully epistatic to ztl, suggesting that another com-ponent is indeed involved in the regulation of CBFexpression. PRR5 is a repressor of the CBF genes(Nakamichi et al., 2012) and also is negatively regulatedby ZTL through ubiquitin-mediated degradation (Kibaet al., 2007). Thus, a higher level of PRR5 in the ztlmutant could be partly responsible for the dramaticdecrease in CBF3 expression in the mutant (Fig. 6D).The prr5 single mutant also displayed a phenotypesimilar to hy5, with significantly higher CBF3 andCOR15a expression levels compared with the wild type

Figure 6. ZTL is involved in the regulation of CBF3 expression. A, Plants were grown in short-day conditions (9/15 h light/dark),and 3-week-old plants of Col and crd were analyzed for ZTL protein levels every 3 h starting at dawn and the last samples werecollected 3 h into the dark period. Representatives of three blots from three individual experiments are shown. B, Relative ZTLprotein levels in Col and crd. Protein levels in Col were set as 100% for each blot, and protein levels in crd were compared withCol at the same time point and adjusted according to the a-tubulin loading control levels. Means of the relative levels in threeindependent experiments are presented. ZTL protein levelswere significantly lower in crd comparedwith Col at all time points, asdemonstrated by Student’s t test: *, P, 0.05; **, P, 0.01; and ***, P, 0.001. C, Co-IP interaction was identified between ZTLand HY5 protein using Arabidopsis protoplasts. The blots presented are representative of three individual experiments. Theimages of negative controls and expressed ZTL and HY5 proteins are cut from the same blot, as indicated by the center lines, andmerged for the illustration. D, Plants were grown in short-day conditions, and 3-week-old plants of Col, crd, hy5, prr5-1, ztl-3,ZTL-OX, ztl crd, ztl hy5, and ztl prr5 were analyzed for CBF3 (At4g25480) expression 6 h into the light period. Gene expressionwas normalized to ubiquitin-like protein (UBI; At4g36800) and related to the amount present in wild-type Col. Each data pointrepresents the mean6 SD of at least three biological replicates. CBF3 expression was significantly different in crd, hy5, prr5, ztl,and ZTL-OX comparedwith Col and in ztl hy5 and ztl prr5 compared with ztl, as demonstrated by Student’s t test: ***, P, 0.001.








(Figs. 6D and 7A). In addition, similar to hy5, the prr5mutant was less sensitive to the GDA treatment withregard to CBF3 expression (Supplemental Fig. S3E).However, in contrast to hy5, CBF1 and CBF2 expressionwas higher compared with the wild type in the prr5mutant (Supplemental Fig. S4). The prr5 ztl doublemutant displayed higher CBF3 and COR15a expressionlevels compared with the ztl single mutant, but again,prr5 is not fully epistatic to ztl, suggesting that HY5 andPRR5 act in concert to repress CBF3 expression in re-sponse to the plastid signal.

The protein levels of COR15a also were investigated(Fig. 7B), and protein abundance in thewild type and themutants correlated with the observed gene expressionprofiles, with the high amounts of COR15a protein in thehy5 and prr5mutants and low amounts in all other singlemutants compared with the wild type. However, ahigher amount of COR15a protein was found in the hy5crd, hy5 ztl, and prr5 ztl double mutants compared withthe crd and ztl single mutants (Fig. 7B). Taken together,these results indicate that HSP90 and ZTL, together withHY5 and PRR5, respond to the plastid signal and regu-late CBF3 and downstream COR15a expression duringphotoperiodic conditions.

HY5, a Putative Target for ZTL-Mediated Degradation

ZTL and HY5 were shown to interact physically invivo (Fig. 6C). ZTL is responsible for the proteasome-dependent degradation of TOC1 and PRR5 (Más et al.,2003; Kiba et al., 2007). Possibly, ZTL also targets HY5for degradation. HY5 protein levels were determined inwild-type, ztl, and ZTL-OX plants during the light pe-riod and 3 h after dusk. The results from three trialsindicated possibly higher HY5 protein levels in the ztlmutant and suggested slightly lowerHY5 protein levelsin the ZTL-OX line compared with the wild type(Supplemental Fig. S7C). To test the possibility that thedegradation of HY5 may be mediated by ZTL, wemeasured HY5 half-life under white light in etiolatedseedlings of the wild type, ztl, and ZTL-OX (Somerset al., 2004). We followed the protocol to determineprotein half-life using cycloheximide (CHX) describedby Kiba et al. (2007), where the seedlings were exposedto white light for 11 h and then treated with CHX andincubated under light conditions. In parallel to the CHXtreatment, a mock experiment was performed. Sampleswere collected and HY5 levels were detected (Fig. 8A;Supplemental Fig. S7D). In the mock samples, nochange in the HY5 protein level was found during thetime period for any of the genotypes (Supplemental Fig.S7D). The level of HY5 protein was reduced signifi-cantly compared with the control at 9 and 12 h after theCHX treatment in wild-type plants, suggesting somelevel of degradation (Fig. 8B). In ztl, the HY5 half-lifewas extended and the protein levels were almost un-changed during the entire experiment (Fig. 8). In con-trast, the degradation of HY5 was enhanced slightly inthe ZTL-OX line (Fig. 8), where the level of HY5 proteinwas reduced significantly compared with the control at 6,9, and 12 h after the CHX treatment. These results aresimilar to what was seen for PRR5 (Kiba et al., 2007), butthe effects of variation in ZTL levels on HY5 turnover aremuch less impactful, suggesting that ZTLmaybe only oneof multiple factors in the regulation of HY5 protein levels.

DISCUSSION

We describe a mechanism where retrograde signalstriggered by diurnal oscillations in tetrapyrrole levels

Figure 7. HSP90, ZTL, and HY5 regulate COR15a in response to theplastid signal. A, Plants were grown in short-day conditions (9/15 hlight/dark), and 3-week-old-plants were analyzed for COR15a(At2g42540) expression 9 h into the light period. Gene expression datawere normalized to ubiquitin-like protein (UBI; At4g36800) and relatedto the amount present in wild-type Col. Each data point represents themean 6 SD of at least three biological replicates. COR15a expressionwas significantly different in crd, hy5, Col-hsp90-1, Col-hsp90-3, ztl,and prr5 compared with Col, in hy5 crd compared with crd, and in ztlcrd, ztl hy5, and ztl prr5 compared with ztl. Statistical differences werecalculated using Student’s t test: *, P , 0.05; and ***, P , 0.001. B,COR15a protein levels were analyzed 9 h into the light period.Coomassie Blue-stained gels are shown for loading controls. Theblots are representative from at least three individual experimentswith the same relative protein levels.


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converge with the circadian clock to fine-tune nucleargene expression under photoperiodic conditions. CBFexpression is under circadian control, and the oscilla-tion of CBF expression peaks 6 h into the light period(Fig. 2; Supplemental Fig. S1). Accumulation of thetetrapyrroles, Mg-ProtoIX and Mg-ProtoIX-ME, alsoshowed a strong rhythmic pattern where the tetrapyr-role levels increased rapidly at the beginning of the lightperiod and thereafter decreased until dusk (Fig. 2;Papenbrock et al., 1999). The rhythmic patterns of tet-rapyrrole accumulation and CBF expression indicatethat, when the tetrapyrrole levels peak, a repression ofCBF expression is triggered (Fig. 2). This is supportedby the results with the tetrapyrrole-overaccumulatingmutant crd, which showed the same rhythmic circadianpattern of CBF expression as the wild type but withstrongly repressed CBF expression levels. The diurnaloscillations of Mg-ProtoIX and Mg-ProtoIX-ME arecompletely abolished when plants are transferred toconstant light (Fig. 2), and the activity of enzymes re-sponsible for tetrapyrrole biosynthesis is inhibited whenplants are exposed to continuous light (Papenbrock et al.,1999). Thus, the accumulation of Mg-ProtoIX and Mg-ProtoIX-ME is not regulated by any clock-controlledmechanisms and requires daily light and dark changesin order to maintain the oscillation pattern. When theplants were exposed to constant light, the circadianregulation of CBF3 expression was maintained in boththe wild type and crd, but the observed difference in

expression levels under diurnal conditions between thewild type and crd was abolished (Fig. 2). Thus, underconstant light conditions, the role of the tetrapyrrole-triggered plastid signal is diminished and does notcontribute to the regulation of CBF3, which then wasmaintained only by the circadian clock components.

Several possible mechanisms have been proposed toexplain the regulation of CBF expression under warmgrowth conditions, including activation by LHY andCCA1 (Espinoza et al., 2010; Dong et al., 2011) andinhibition by PRR5, PRR7 and PRR9 (Nakamichi et al.,2009). Overexpression of CBF3 has been shown to af-fect vegetative growth and flowering time (Liu et al.,1998; Kasuga et al., 1999; Gilmour et al., 2000), sug-gesting that CBF3 is involved in processes other thanthe response to low temperatures. This is further sup-ported by the downstream COR genes that respond notonly to cold; COR15a expression, for example, also isaltered in plants with defective chloroplasts and alteredlevels of tetrapyrroles (Nakayama et al., 2007; Bang et al.,2008; Dong et al., 2011). The promoters of CBF1 andCBF2 have G-box elements close to the transcriptionalstart, and the transcription factor PIF7 has been reportedto bind to the G-box element inCBF2 and to function as arepressor of CBF1 and CBF2 expression under circadiancontrol (Kidokoro et al., 2009). However, instead ofa conserved G-box element, CBF3 contains Z-box ele-ments close to the transcriptional start. Both the G-boxand Z-box elements respond to light signals and have

Figure 8. HY5 protein is potentially subject to ZTL-mediated degradation. A, Five-day-old etiolated plants were exposed towhitelight for 11 h and then incubated with 100 mM CHX. Samples were collected at the indicated time points and analyzed for HY5protein levels. a-Tubulin (a-TUB) was used as a loading control. Three representative blots from individual experiments areshown. B, Quantification of the results obtained in A. Each data point was normalized to the loading control levels and related tothe amount present at ZT 0 for each genotype. Data points represent means 6 SD of at least three independent blots and ex-periments. HY5 protein levels were significantly lower in wild-type (WT) Col after 9 and 12 h of incubation comparedwith Col at0 h and in ZTL-OX after 6, 9, and 12 h of incubation compared with ZTL-OX at 0 h, as demonstrated by Student’s t test: **, P ,0.01; and ***, P , 0.001.






been shown to contain the core ACGT enriched in genesresponding to the plastid signal triggered by tetrapyrroleaccumulation (Strand et al., 2003). HY5 was describedpreviously to respond to plastid signals (Ruckle et al.,2007; Kindgren et al., 2012), and in the experiments wereport here, HY5 was confirmed to bind to the promoterfragments of CBF1, CBF2, COR15a, and CBF3, but withthe strongest affinity for CBF3 (Fig. 3). In support of thebiochemical data from the EMSA studies, the hy5mutantshowed higherCBF3 expression comparedwith thewildtype, whileCBF1 andCBF2 expression in the hy5mutantwas similar to that in the wild type (Fig. 4; SupplementalFig. S4). Thus, the preferred binding of HY5 to the CBF3promoter was shown in vivo, where HY5 functions as arepressor of CBF3 expression, but the interaction of HY5to the promoters of CBF1 and CBF2 in vitro has no orlittle effect on CBF1 and CBF2 expression in vivo. Fur-thermore, the hy5 crd double mutant released the strongrepression of CBF3 and COR15a expression shown bythe crd single mutant (Figs. 4 and 7). Greater abundanceof the COR15a protein also was found in both hy5 andhy5 crd mutants compared with the wild type (Fig. 7).Similar to what was observed for crd, the difference inCBF3 expression under diurnal conditions between thewild type and hy5 was abolished when the plants wereexposed to constant light conditions, suggesting thatHY5 responds to the plastid signal triggered by the di-urnal changes in tetrapyrrole levels (Fig. 4). Thus, therepression of CBF3 expression in response to the plastidsignal triggered in the crd mutant most likely involvesHY5.However, the analysis of the double hy5 crdmutantsuggested that other components must be involved inthe regulation of CBF3 expression. PRR5 was shown tobe a repressor of the CBF genes (Nakamichi et al., 2012),and similar to the hy5mutant, prr5 showed higher CBF3expression compared with the wild type. In contrast tohy5, CBF1 and CBF2 expression also was higher com-pared with the wild type in prr5 (Fig. 6; SupplementalFig. S4).

HY5 was shown to be part of a regulatory systemincluding HSP90 proteins, which are modified by the

accumulation of tetrapyrroles in response to oxidativestress (Kindgren et al., 2012). Oxidative stress results inreduced flux through the tetrapyrrole biosyntheticpathway and a significant accumulation of the chloro-phyll intermediates Mg-ProtoIX and Mg-ProtoIX-ME(Stenbaek et al., 2008; Kindgren et al., 2011; Zhang et al.,2011). Reduced flux through the tetrapyrrole pathwayand the accumulation of Mg-ProtoIX were shown toinhibit the ATPase activity of HSP90, which in turnresulted in reduced expression of PhANGs via HY5(Kindgren et al., 2012). CBF3 and COR15a expressionalso is dependent on functional HSP90, and both ex-pression levels and protein content were reducedcompared with the wild type in two independentHSP90 RNAi lines (Figs. 5 and 7). In addition, treatmentwith the inhibitor of HSP90 GDA resulted in a clearrepression of CBF3 expression in the wild type and crd,confirming that HSP90 activity is involved in the reg-ulation of CBF3 expression (Fig. 5B). In the hy5 and prr5mutants, CBF3 expression was insensitive to GDAtreatment, supporting that HY5 and PRR5 act down-stream of HSP90. However, CBF1 and CBF2 expressionwas repressed similar to that in the wild type in hy5following GDA treatment (Supplemental Fig. S3),which argues for a specific regulation of CBF3 by HY5.

Recently, it also was demonstrated that HSP90 is in-volved in maturation of the circadian clock-associatedF-box protein ZTL (Kim et al., 2011). Treatment with theinhibitor GDA or RNAi-mediated depletion of cytosolicHSP90 reduced the levels of ZTL and lengthened thecircadian period, consistent with ztl loss-of-function al-leles (Kim et al., 2011). Thus, maturation of ZTL byHSP90was shown to be essential for the proper functionof the circadian clock. With F-box proteins such as ZTLas clients, HSP90 has a unique and central role in pro-teostasis and thereby could control many different cel-lular responses. Similar to crd and theHSP90 RNAi lines,ztl displayed lower expression levels for CBF3 andCOR15a compared with the wild type (Figs. 6 and 7).Furthermore, CBF3 expression was similar in the ztl crddouble mutant compared with the ztl single mutant

Figure 9. Model for the regulation of CBF expression under photoperiodic conditions. We propose a model for integrationbetween plastid and circadian signaling pathways. HSP90 responds to the plastid signal triggered by tetrapyrrole accumulation,and its activity is inhibited. The accumulation of tetrapyrroles demonstrated a strong rhythmic pattern, and when the tetrapyrrolelevels are elevated, HSP90 is inactivated. A consequence of HSP90 inactivation is a significant reduction in ZTL levels. Low levelsof ZTL would result in increased levels of HY5 and PRR5 and a repression of CBFs. HY5 and PRR5 act in concert to repress CBF3and PRR5 represses CBF1 and CBF2. The plastid signal thereby contributes to the rhythm of CBF and COR expression during day/night cycles.


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(Fig. 6D). Thus, no enhanced suppression of expressioncould be found in the double mutant compared withthe ztl single mutant, supporting the suggestion thatCRD and ZTL are genetically linked and that ZTL isdownstream of tetrapyrrole accumulation. In addition,the protein levels of ZTL were significantly lower at alltime points investigated in the crd mutant comparedwith the wild type (Fig. 6), supporting the demonstratedinhibition of HSP90 activity by accumulated tetrapyr-roles (Kindgren et al., 2012). It is clear that both HY5 andZTL are linked toHSP90 function. In addition, in a Co-IPassay using Arabidopsis protoplasts, HY5 was foundto interact directly with ZTL (Fig. 6), emphasizingthe connection between these two components. TheF-box protein ZTL is responsible for the proteasome-dependent degradation of TOC1 and PRR5 (Más et al.,2003; Kiba et al., 2007). It is clear that PRR5 is involved inthe regulation of CBF expression during photoperiodicgrowth (Figs. 6 and 7). In addition, similar to PRR5, in-creased stability of HY5 was seen in the ztl mutant,suggesting that HY5 possibly is a target for ZTL-mediated degradation. Thus, the effects seen in ztl withseverely repressed expression of CBF3 could be a re-sponse to higher levels of HY5 and PRR5. It is unlikelythat TOC1 would contribute to CBF regulation, since noeffect onCBF1 toCBF3 expressionwas reported in the tocmutants. Putative target genes for TOC1, however, doinclude HY5 (Huang et al., 2012), but HY5 was not dif-ferentially expressed in overexpressors of TOC1 or in thetoc1-2mutant (Legnaioli et al., 2009).HY5was not foundto be regulated by PRR5 (Nakamichi et al., 2012), indi-cating that a direct effect of PRR5 on HY5 levels also isunlikely in the ztl mutant. Thus, CBF expression is reg-ulated by a combination of HY5 and PRR5 during pho-toperiodic growth.The regulation of ZTL by the plastid signal via

HSP90 is most likely the site of interaction between theclock and the plastid signaling pathways. In additionto the impaired retrograde signaling, the crd mutanthas a pale phenotype and reduced growth (Totteyet al., 2003; Bang et al., 2008). Thus, it is possible thatthe plastid signal triggered in the crd mutant could bemore complex than the described overaccumulation oftetrapyrroles. However, connections between plastidand circadian signaling pathways have been suggestedin the literature, and mutations in CHLOROPLASTRNA BINDING (CRB) resulted in an increased am-plitude of expression of CCA1 and LHY. The crbplants showed altered chloroplast morphology andimpaired chlorophyll biosynthesis, suggesting an in-volvement of the chloroplast, and tetrapyrrole bio-synthesis in particular, in the regulation of thecircadian clock in Arabidopsis (Hassidim et al., 2007).It has been shown that CCA1 and LHY bind to thepromoters of CBF1 to CBF3 to activate their tran-scription (Dong et al., 2011). In addition, CCA1 andHY5 together positively regulate LHCB gene expres-sion (Andronis et al., 2008). Hence, it is possible thatHY5 impacts the CCA1 regulation of CBFs, positivelyor negatively depending on the chloroplast signaling

status. PRR5 and PIF7 could potentially interact asrepressors of CBF1 and CBF2 expression. However,the difference in CBF1 and CBF2 expression betweenthe wild type and the pif7 mutant was only foundwhen the plants were exposed to constant light; underlight/dark cycles, no difference in CBF expressioncould be detected (Kidokoro et al., 2009; Lee andThomashow, 2012).

We propose here a model for integration betweenplastid and circadian signaling pathways (Fig. 9). Thiscross talk would provide a mechanism for fine-tuninggene expression during light/dark cycles. It was sug-gested that HSP90 activity is under diurnal or circadiancontrol (Kimet al., 2011), andwehave shown thatHSP90responds to a plastid signal triggered by the accumula-tion of tetrapyrroles (Kindgren et al., 2012). Accumula-tion of tetrapyrroles demonstrated a strong rhythmicpattern (Fig. 2) and thus could explain the phase-specificdifferences in HSP90 activity (Kim et al., 2011). Whentetrapyrrole levels are elevated, HSP90 is inactivated,and as a consequence, there is a significant reduction inZTL levels (Fig. 6). Low levels of ZTL would potentiallyresult in increased protein levels of HY5 and PRR5 and arepression of CBFs (Fig. 9). HY5 and PRR5 act in concertto repress CBF3, and PRR5 represses CBF1 and CBF2.The plastid signal thereby contributes to the rhythm ofCBF expression during day/night cycles. These findingsprovide a mechanism by which plastid signals convergewith and impact on the activity of well-defined clockcomponents involved in circadian regulation, and ourresults provide further evidence that the functional stateof the chloroplast is an important factor that affectsthe circadian system. The close interaction between thecircadian clock and chloroplast retrograde signaling sys-tems could fine-tune photosynthetic activity under pho-toperiodic conditions.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) plants were grown for 3 weeks on soil inshort-day conditions (9/15-h light/dark and 22°C/18°C) with a light intensity of150mmol photonsm22 s21. Allmutantswere in theCol backgroundanddescribedelsewhere: crd1 (Ankele et al., 2007), hy5-1 (Maxwell et al., 2003;Kleine et al., 2007),Col-hsp90-RNAi lines (Kindgren et al., 2012), ztl-3 (Jarillo et al., 2001), and prr5-1(Eriksson et al., 2003). For the constant light experiments, plants were kept inconstant 150 mmol photons m22 s21 light and 22°C. For the Mg-ProtoIX feedingexperiments, plants were grown on soil in short-day conditions for 3 weeks andthen transferred to either 13MS or 13MS + 50mMMg-ProtoIX solution and keptin continuous low light (20 mmol photonsm22 s21). Roots were rinsedwith waterbefore incubation. The feeding was started at 9 AM, and plants were sampled 12 hlater. To avoid contamination of Mg-ProtoIX in solution, only green tissue wassampled for real-time PCR and HPLC analysis. Cold treatment was performed in4°C, short-day conditions (9/15 h), and 150mmol photonsm22 s21 light. Plants forGDA feeding were grown on 13 MS + 1% Suc plates for 10 d in short-day con-ditions and then incubated with 13MS or 13MS + 80 mM GDA + 0.01% TritonX-100 solution for 24 h. The feeding was started at ZT 6, and fresh GDA solutionwas added after 3 and 18 h. For the CHXexperiment, seedswere plated on 13MSplates, exposed to 3 h of 150 mmol photons m22 s21 light, and then grown in thedark for 5d. Etiolatedplantswere exposed to 11hof 150mmolphotonsm22 s21 lightbefore starting the incubation with 100 mM CHX solution in 13MS + 0.01% TritonX-100 or mock treatment with 13MS + 0.01% Triton X-100. Plants were incubatedshaking at 100 rpm for 12 h.





RNA Isolation, Complementary DNA Synthesis, and Real-Time PCR

Total RNA was isolated using the EZNA Plant RNA mini kit, and DNasetreatment was performed using Thermo Scientific DNase I, RNase-freeaccording to the manufacturer’s instructions. Using the iScript cDNA Synthe-sis Kit (Bio-Rad), complementary DNA (cDNA) was synthesized from 0.5 mg oftotal RNA according to the manufacturer’s instructions. cDNA was diluted10-fold, and 3 mL of the diluted cDNA was used in a 10-mL iQ SYBR GreenSupermix reaction (Bio-Rad). All reactions were performed in three technicalreplicates using primers for the genes COR15a, COR47, CBF1, CBF2, CBF3,FER1, GST5, andMAPK18, and relative gene expression was normalized to theexpression level of ubiquitin-like protein (Supplemental Table S1). Quantitativereal time-PCR was run in the CFX96 real-time system (Bio-Rad) and monitoredusing the CFX Manager (Bio-Rad). The adjustment of baseline and thresholdwas done according to the manufacturer’s recommendations. Data were ana-lyzed using LinRegPCR (Pfaffl, 2001; Ramakers et al., 2003), and the relativeabundance of all transcripts amplified was normalized to the constitutive ex-pression level of ubiquitin-like protein.

HPLC Analysis

HPLC analyses were done according to the method described byMochizukiet al. (2008). Leafmaterial was homogenized in acetone:0.1 MNH4OH (9:1, v/v).Column eluent was monitored by UV light detection, and tetrapyrroles wereidentified and quantified using authentic standards. Mg-ProtoIX and Mg-ProtoIX-ME were purchased from Frontier Scientific.

Expression and Purification of HY5

A fragment of the coding region of HY5 was amplified from Arabidopsis(Col) genomic DNA (Supplemental Table S1). The fragment was cloned intopDONR207 using Gateway technology (Invitrogen) according to the manu-facturer’s instructions. The cloned fragment was sequenced to confirm that nomistakes were made during the PCR. For expression, the fragment was sub-sequently cloned into pETG-10K vector, creating a His-HY5 fusion protein. Theprotein was expressed in Escherichia coli Rosetta2 cells. Transformed cells weregrown in Luria-Bertani medium at 37°C until the optical density at 600 nmreached 0.4 and transferred to 16°C for 30 min. Expression was induced byadding isopropylthio-b-galactoside to a final concentration of 0.2 mM. The in-duced culture was grown at 16°C for 4 h and harvested. The pellet was dis-solved in ice-cold lysis buffer (50 mM Tris, pH 7.2, 500 mM NaCl, 10 mM

imidazole, and 7 mM b-mercaptoethanol), and cells were destroyed by ho-mogenization. After centrifugation (13,000g, 15 min, and 4°C), the supernatantwas loaded onto a nickel-nitrilotriacetic acid agarose column (Bio-Rad). Theprotein was purified according to the manufacturer’s instructions. Elutedprotein was dialyzed overnight at 4°C into dialysis buffer (50 mM Tris, pH 7.2,500 mM NaCl, 50% glycerol, and 7 mM b-mercaptoethanol).

EMSA

EMSAs were done according to Schallenberg-Rüdinger et al. (2013). Briefly,purified proteinwas incubatedwith 500 pM labeledDNAoligonucleotide (Sigma)for 30min at 25°C. Final binding reactions included 10%glycerol, 13THE, pH7.2,200 mM NaCl, 5 mM dithiothreitol, 0.1 mg mL21 bovine serum albumin,0.5 mg mL21 heparin, and 8 units of RNAseOUT. Samples were loaded onto aprerun 5% 13 THE native polyacrylamide gel, and a constant voltage of 100 Vwas applied. Gels were visualized in a Typhoon scanner (GE Healthcare).

Western-Blot Analysis

To determine the levels of COR15a protein in the plants, 3-week-old plantsgrown in short-day conditions were sampled 9 h into the light period. A total of20 mg of ground plant material was used for protein extraction with 200 mL ofextraction buffer (10% [w/v] SDS, 20% [v/v] glycerol, 0.2 M Tris-HCl, pH 6.8,0.05% [w/v] Bromophenol Blue, 10 mM b-methanol, and 5 mM dithiothreitol).The samples were loaded onto a 16% gel and separated by SDS-PAGE. Equalprotein loading was determined by Coomassie Blue staining, and the COR15aantibody (Nakayama et al., 2007) was used for detection by western blotting.For ZTL protein levels, plants were grown for 3 weeks in short-day conditionsand sampled 0, 3, 6, and 9 h into the light period and also 3 h into the dark

period. The samples were prepared as described above and separated on a 12%gel, and ZTL antibody (Kim et al., 2011) was used for detection by western blot-ting. To determine HY5 protein levels in Col and ztl during short-day conditions,plantswere grown for 7 d onMSmedium+ 1%Suc plates and sampled 0, 3, 6, and9 h into the light period and also 3 h into the dark period. The samples wereprepared as described above and separated onto a 12% gel, and two differentHY5antibodies (Santa Cruz Biotechnology and a gift from Dr. X.W. Deng) were usedfor detection by western blotting. In contrast to the western blot presented byKleine et al. (2007) using the Santa Cruz Biotechnology HY5 antibody, whereseedlings were used, we used mature plants. In the mature plants, the hy5-1mu-tant contains someHY5protein, demonstrating that it is not a complete null allele.Equal protein loading was determined by the detection of a-tubulin levels on thesame membrane as the protein of interest. Quantification of the ZTL and HY5protein levels was done using the program ImageJ, and each data point wasnormalized to the loading control levels on each blot.

Co-IP Assay

Expression constructs were generated for the assay by cloning full-lengthcoding sequences of ZTL andHY5 into pRT104_3Myc and pRT104_3HAvector,respectively. Arabidopsis Landsberg cell cultureswere grown at constant 12/12-hlight/dark, 23°C/23°C, with a light intensity of 150 mmol m22 s21. Protoplastswere isolated and transformed with ZTL-Myc or HY5-HA constructs and an-alyzed as described by Shaikhali et al. (2012).

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Expression of CBF1 and CBF2 in Col and crd.

Supplemental Figure S2. Tetrapyrrole contents in 3-week-old plants fol-lowing a shift to 4°C.

Supplemental Figure S3. Tetrapyrrole levels following Mg-ProtoIXfeeding.

Supplemental Figure S4. Expression analysis of CBF1 to CBF3 in Col, hy5,prr5, and ztl.

Supplemental Figure S5. ZTL protein contents in two independent HSP90RNAi lines.

Supplemental Figure S6. Expression analysis of CBF3 and COR15 during aday/night cycle in Col and ztl.

Supplemental Figure S7. HY5 protein contents in Col, ztl, and ZTL-OXunder control conditions.

Supplemental Table S1. Primers used in real-time PCR analysis and forcDNA synthesis.

ACKNOWLEDGMENTS

We thank Dr. Takehito Inaba for the COR15am antibodies, Dr. DavidSomers for the ZTL antibody and the ZTL-OX line, Dr. Xing Wang Deng forthe HY5 antibody, Dr. Alex Webb for the gift of the ztl-3mutant, Dr. Andrew J.Millar for the prr5-1 mutant, Dr. Manuela Jurca for help with generating theprr5-1 ztl-3 double mutant, and Dr. Carole Dubreuil for help with the westernblots.

Received March 8, 2016; accepted April 9, 2016; published April 14, 2016.

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