genome-wide gene expression analysis reveals a critical role … · genome-wide gene expression...

Genome-Wide Gene Expression Analysis Reveals aCritical Role for CRYPTOCHROME1 in the Responseof Arabidopsis to High Irradiance1[W]

Tatjana Kleine2, Peter Kindgren, Catherine Benedict, Luke Hendrickson, and Åsa Strand*

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

Exposure to high irradiance results in dramatic changes in nuclear gene expression in plants. However, little is known aboutthe mechanisms by which changes in irradiance are sensed and how the information is transduced to the nucleus to initiate thegenetic response. To investigate whether the photoreceptors are involved in the response to high irradiance, we analyzedexpression of EARLY LIGHT-INDUCIBLE PROTEIN1 (ELIP1), ELIP2, ASCORBATE PEROXIDASE2 (APX2), and LIGHT-HARVESTING CHLOROPHYLL A/B-BINDING PROTEIN2.4 (LHCB2.4) in the phytochrome A (phyA), phyB, cryptochrome1(cry1), and cry2 photoreceptor mutants and long hypocotyl5 (hy5) and HY5 homolog (hyh) transcription factor mutants. Followingexposure to high intensity white light for 3 h (1,000 mmol quanta m22 s21) expression of ELIP1/2 and APX2 was stronglyinduced and LHCB2.4 expression repressed in wild type. The cry1 and hy5 mutants showed specific misregulation of ELIP1/2,and we show that the induction of ELIP1/2 expression is mediated via CRY1 in a blue light intensity-dependent manner.Furthermore, using the Affymetrix Arabidopsis (Arabidopsis thaliana) 24 K Gene-Chip, we showed that 77 of the high light-responsive genes are regulated via CRY1, and 26 of those genes were also HY5 dependent. As a consequence of themisregulation of these genes, the cry1 mutant displayed a high irradiance-sensitive phenotype with significant photoinacti-vation of photosystem II, indicated by reduced maximal fluorescence ratio. Thus, we describe a novel function of CRY1 inmediating plant responses to high irradiances that is essential to the induction of photoprotective mechanisms. This indicatesthat high irradiance can be sensed in a chloroplast-independent manner by a cytosolic/nucleic component.

Light is not only the primary energy source forplants but it also provides them with information tomodulate developmental processes such as seed ger-mination, seedling establishment, phototropism, chloro-plast movement, shade avoidance, circadian rhythms,and flowering time (Fankhauser and Staiger, 2002;Chen et al., 2004). Plants can detect almost all facets oflight, including direction, duration, and wavelengthusing three major classes of photoreceptors: the red/far-red light-absorbing phytochromes, the blue/UV-Alight-absorbing cryptochromes and phototropins, andthe UV-B-sensing UV-B receptors (Chen et al., 2004).These photoreceptors perceive light signals and initi-ate intracellular signaling pathways involving proteo-lytic degradation of signaling components and largereorganization of the transcriptional program to mod-

ulate plant growth and development (Chen et al.,2004).

In photosynthesis, light energy is absorbed by thelight-harvesting antennae and converted into chem-ical energy by the reaction centers. However, whenphoton fluence exceeds the photon utilization capac-ity of the chloroplast, photosynthesis becomes photo-inhibited and the reaction centers, particularly PSII,become irreversibly damaged and require repair (Aroet al., 1993a, 1993b). Furthermore, elevated excitationpressure has been demonstrated to increase the pro-duction of reactive oxygen species (ROS; Karpinskiet al., 1997; Huner et al., 1998; Foyer and Allen, 2003),and the damaging effects of ROS include oxidation oflipids, proteins, and enzymes necessary for properfunction of the chloroplast and the cell as a whole(Foyer and Allen, 2003). To protect themselvesagainst extensive damage, plants have the ability tosense when photon fluence exceeds the photon utili-zation capacity of the chloroplast and communicatethis information to stimulate changes in nuclear andchloroplast gene expression. Recent microarray ex-periments have revealed that expression of a largenumber of nuclear-encoded genes is affected by ex-posure to high irradiance (Rossel et al., 2002; Kimuraet al., 2003; Richly et al., 2003; Vanderauwera et al.,2005). The mechanisms by which excess irradiance issensed and how the information is transduced to thenucleus to initiate a genetic response are unknown,but it is well established that the redox state of the

1 This work was supported by the Swedish Research Foundationand Foundation for Strategic Research (INGVAR grant to Å.S.).

2 Present address: Department of Biology, Ludwig-Maximilians-Universität Munich, D–80638 Munich, Germany.

* Corresponding author; e-mail [email protected];fax 46–90–786–6676.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Åsa Strand ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.107.098293

Plant Physiology, July 2007, Vol. 144, pp. 1391–1406, www.plantphysiol.org � 2007 American Society of Plant Biologists 1391

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plastoquinone electron carrier pool is correlated withthe expression of photosynthetic genes encoded inboth the chloroplast and the nucleus (Escoubaset al., 1995; Huner et al., 1998; Karpinski et al., 1999;Pfannschmidt et al., 1999, 2001; Pfannschmidt, 2003).Furthermore, a unique light- and redox-controlledprotein phosphorylation system has evolved in plantthylakoid membranes where intrinsic protein kinasesare activated by light or reducing conditions and sub-sequently phosphorylate the membrane proteins of PSIIand its light-harvesting antenna, light-harvesting com-plex II (LHCII; Vener et al., 1998). The phosphorylationstate of these proteins has been suggested to be in-volved in the regulation of LHC expression in thenucleus (Rintamaki et al., 1997). In addition, underhigh irradiance conditions where the equilibrium be-tween different ROS (e.g. hydrogen peroxide and OH•)production and scavenging is perturbed, subsequentchanges in concentrations or rates of ROS productioncould also be initiators of signaling pathways originat-ing in the chloroplast (Karpinski et al., 2003; Apel andHirt, 2004).

Despite extensive work, the mechanisms by whichexcess irradiance is sensed and how the information istransduced to the nucleus to initiate a genetic responsehave remained elusive. Are plastid signals alone re-sponsible for the regulation of nuclear gene expressionin response to excess irradiance? The dramatic impacton nuclear gene expression by exposure to high inten-sity white light suggests that several signaling path-ways are involved. Hence, we wanted to test whethercomponents known to control photomorphogenesis suchas cryptochromes (CRY1 and 2), phytochromes (PHYAand B), and two of their downstream transcriptionfactors (LONG HYPOCOTYL5 [HY5] and HY5 HO-MOLOG [HYH]) also are involved in the response toexcess irradiance. We tested the expression of genesencoding EARLY LIGHT-INDUCIBLE PROTEIN1 and 2(ELIP1 and ELIP2), ASCORBATE PEROXIDASE2 (APX2),and LIGHT-HARVESTING CHLOROPHYLL A/B-BIND-ING PROTEIN2.4 (LHCB2.4) after high intensity whitelight treatment in the cry1, cry2, phyA, phyB, phyAphyB,hy5, and hyh mutants. The cry1 and hy5 mutants showedmisregulation of ELIP1/2 in response to high intensitywhite light, and by using the Affymetrix Arabidopsis(Arabidopsis thaliana) 24 K Gene-Chip representing24,000 genes, we could demonstrate that a large groupof the HL-responsive genes were regulated via aCRY1-mediated response and that 26 of those genes

were also HY5 dependent. Our study demonstrates anovel function of CRY1 as a mediator of plant responseto changes in irradiance and provides new insight intothe high light stress-responsive transcriptome.

RESULTS

Exposure to High Light Results in Adaptive Changesof the Transcriptome

Arabidopsis seedlings were grown for 7 d at 100mmol quanta m22 s21 continuous white light (growthlight [GL]) and exposed for 3 h to a high intensitywhite light treatment of 1,000 mmol quanta m22 s21

(HL). Exposure to HL results in significant light stress,as shown by the gradual drop in the variable tomaximal fluorescence ratio (Fv/Fm) following expo-sure (Table I). Long-term exposure (12 h) to HL resultsin a drop in Fv/Fm from 0.83 to 0.59 in wild type,indicative of PSII photoinactivation (Table I). To geta robust gene set responding to these experimentalconditions, wild-type samples from three independentbiological experiments were hybridized to ATH1 Ge-nome Arrays (Affymetrix). Differentially expressedgenes were identified with a combination of logit-t(Lemon et al., 2003) and the Filter on Fold Change toolin GeneSpring 7.3 (Agilent Technologies; Schmid et al.,2003). A total of 992 genes showed more than 2-foldchange in expression in response to 3-h HL treatment.Thus, approximately 4% of all genes represented on thechip demonstrated changes in expression in responseto HL, 660 genes were 2-fold up-regulated, and 332were 2-fold down-regulated (Supplemental Table S1).

Under our experimental conditions, ELIP1 andELIP2 showed a very strong induction of expression,100-fold and 88-fold, respectively, in response to 3-hHL treatment. As described previously, ELIP1 wasmore strongly induced by HL compared to ELIP2(Heddad et al., 2006). APX2 was induced 6-fold andLHCB2.4 was reduced to less than 25% of the control.The ELIP1, ELIP2, APX2, and LHCB2.4 genes all dem-onstrated a robust response to our HL exposure andthey all encode key components of photosynthetic lightstress response. In addition, the observed response toHL of these genes is well documented in plants exposedto somewhat different experimental, high irradianceconditions (Adamska et al., 1992; Karpinski et al., 1999;Kimura et al., 2001; Rossel et al., 2002; Vanderauweraet al., 2005; Heddad et al., 2006). Thus, these four genes

Table I. Maximum quantum efficiency of PSII (Fv /Fm) in wild type, cry1, and hy5

Plants were grown for 4 to 5 weeks, and Fv/Fm was estimated before and after exposure to 3, 6, and 12 h ofwhite light at 1,000 mmol of photons m22 s21. Measurements were made in humidified air. Each point is themean 6 SE of four to six leaves from four to six individual plants after 60-min dark acclimation.

Genotype Control 3 h 6 h 12 h

Wild type 0.829 6 0.003 0.728 6 0.012 0.657 6 0.064 0.593 6 0.003cry1 0.822 6 0.002 0.682 6 0.026 0.438 6 0.037 0.314 6 0.022hy5 0.824 6 0.005 0.710 6 0.014 0.661 6 0.059 0.602 6 0.008

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were selected as marker genes for the HL response inour further analysis using the photoreceptor mutantscry1, cry2, phyA, phyB, and phyAphyB and the tran-scription factor mutants hy5 and hyh.

The Role of Photoreceptors in the Response to

High Irradiance

To test a possible role of the photoreceptors CRY1,CRY2, PHYA, and PHYB in the response to high irra-diance, we analyzed ELIP1/2, APX2, and LHCB2.4 ex-pression in the cryptochrome mutants cry1 and cry2 andthe phytochrome mutants phyA, phyB, and phyAphyB.Seven-day-old wild-type and mutant seedlings grownin continuous white light (100 mmol quanta m22 s21)were exposed to high irradiance conditions identical tothe conditions used for the microarray experiment(HL). In wild type, HL exposure resulted in a stronginduction of ELIP1 (55-fold) and ELIP2 (50-fold), re-spectively (Fig. 1, A and B). Thus, the strong inductionobserved in the microarray experiment was confirmedby real-time PCR. In the cry2 and phyA mutants, theinduction of ELIP1 and ELIP2 expression was similarto that in wild type, whereas in the phyB and phyAphyBmutants, the induction of ELIP1 expression was some-what lower than that in wild type (Fig. 1, A and B). Incontrast, the cry1 mutant showed a strongly reducedinduction of both ELIP1 (11-fold) and ELIP2 (8-fold)relative to wild type (55- and 50-fold, respectively; Fig.1, A and B).

The basic Leu zipper transcription factor HY5 isinvolved in the promotion of light-induced gene ex-

pression (Ang et al., 1998; Chattopadhyay et al., 1998).ELIP1 and ELIP2 expression levels were investigatedin the hy5 mutant and in a T-DNA knockout line of theHY5 homolog HYH, hyh. In the hy5 mutant, both ELIP1and ELIP2 induction was suppressed to levels similarto those observed in the cry1 mutant, whereas the hyhmutant demonstrated wild-type induction (Fig. 1, Aand B). APX2 was induced in all investigated mutantsafter HL treatment (Fig. 1C). LHCB2.4 expression inthe wild type was reduced to 25% of the control levelafter HL treatment (Fig. 1D) and similarly reduced inall investigated mutants. Thus, our results demon-strate that a significant part of the induction of ELIP1and ELIP2 in response to high irradiance is mediatedvia CRY1 and HY5. The misregulation of ELIP1 andELIP2 in response to HL exposure observed in the cry1and hy5 seedlings was also observed in 5-week-oldplants (data not shown).

ELIP1/2 Expression Is Strongly Induced by HighIntensity Blue Light

The mutant analysis demonstrated that the bluelight receptor CRY1 is involved in the HL-inducedexpression of ELIP1 and ELIP2. Consequently, weanalyzed the response of ELIP1/2 in different intensi-ties of blue light (400–540 nm). Wild-type seedlingswere grown for 7 d in continuous white light of100 mmol quanta m22 s21 (10 mmol quanta m22 s21

blue light) and were subjected to 3 h of 25, 50, 100, and200 mmol quanta m22 s21 blue light. ELIP1 and ELIP2 in-duction increased gradually with increasing intensities

Figure 1. Real-time analysis of high light-regulated genes in the cry1, cry2, phyA, phyB, phyAphyB, hy5, and hyh mutants. Wild-type and the different mutant seedlings were grown for 7 d in continuous white light at 23�C and shifted to HL. Expression ofELIP1 (A; At3g22840), ELIP2 (B; At4g14690), APX2 (C; APX1b, At3g09640), and LHCB2.4 (D; At3g27690) was analyzed usingreal-time PCR. The fold induction after HL exposure related to the control of each genotype is presented. The results werenormalized to the expression level of At4g36800 encoding a ubiquitin-protein ligase-like protein. The mean 6 SE of at least fourbiological replicates is shown.

CRYPTOCHROME1 Mediates Plant Responses to High Irradiances

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of blue light (Fig. 2). Following an increase in irradi-ance from 10 to 25 mmol quanta m22 s21, ELIP1 andELIP2 were induced 3- and 3.5-fold, respectively, andwhen transferred from 10 to 200 mmol quanta m22 s21

blue light (BL), ELIP1 and ELIP2 induction increasedto 27- and 37-fold, respectively. Our results supportpublished data using etiolated seedlings demonstratinga blue light intensity-dependent accumulation of ELIPtranscript (Adamska, 1995). However, after transfer ofthe cry1 mutant from 10 to 200 mmol quanta m22 s21

BL, ELIP1 was only 3-fold induced, and the ELIP2induction was abolished. ELIP1 and ELIP2 expressionwas not induced by high intensity red light in wildtype (300 mmol quanta m22 s21; data not shown). Takentogether, these data indicate that the HL-induced ex-pression of ELIP1/2 is predominantly mediated viaCRY1 in a blue light intensity-dependent manner.

CRY1 and HY5 Regulate a Large Number of Genes in

Response to High Light

To determine the identities of the CRY1 and HY5regulons in response to high irradiance, we performedexpression profile analysis of cry1 and hy5 mutants inresponse to HL to be compared with the expressionprofile of wild type exposed to HL. In addition, weanalyzed the expression profile analysis of wild typein response to BL. Seedlings were grown at 100 mmolquanta m22 s21 continuous white light and then sub-jected to BL (wild type) or to HL for 3 h (wild type,cry1, and hy5). Samples from three independent bio-logical experiments were hybridized to ATH1 GenomeArrays (Affymetrix). Wild-type, cry1, and hy5 seed-lings grown and kept at 100 mmol quanta m22 s21

continuous white light (GL) were used as controls.Analysis of the control, GL-grown seedlings demon-

strated that 48 genes were differentially expressed inthe CRY1-deficient seedlings (Supplemental Table S3),

and 290 genes were differently expressed in the HY5-deficient seedlings (Supplemental Table S4) comparedto wild type. Expression analysis of the cry1 and hy5mutants during early light development or following ashift from darkness to light has been performed pre-viously (Ma et al., 2001; Holm et al., 2002; Folta et al.,2003; Jiao et al., 2003; Ohgishi et al., 2004; Ulm et al.,2004). However, a direct comparison between our ex-periment and published results is difficult due to theusage of very different plant material. The 48 genesdifferentially expressed in the cry1 mutant comparedto wild type under our growth conditions were clas-sified into different categories according to The Arabi-dopsis Information Resource (TAIR) gene ontology(http://www.arabidopsis.org/tools/bulk/go/index.jsp).A large proportion of those genes encoded proteinswith unknown function. Three genes encoding redox-related proteins, peroxidase (At2g41480), an electroncarrier (At5g44440), and glutathione dehydrogenase(At1g75270), were identified as genes misregulated inthe cry1 mutant. Only three of the 48 genes differentlyexpressed in the cry1 mutant encode transcription fac-tors (At1g75240, At2g33860, and At5g60450; Supple-mental Table S3).

In the hy5 mutant, as many as 290 genes (SupplementalTable S4) were differentially expressed compared towild type. Among these 290 genes were 14 transcriptionfactors (At5g25190, At5g39860, At1g52830, At5g53980,At1g21910, At2g14210, At3g15540, At3g58120, At1g35560,AT5g25810, At2g47460, At2g17040, and At5g07690)and HY5 (At5g11260) was one of them (SupplementalTable S4). Several genes encoding enzymes in thephenylpropanoid pathway, CHALCONE SYNTHASE(CHS), FLAVANONE 3-HYDROXYLASE (F3H), andFLAVONOL SYNTHASE1 (FLS1), were differently ex-pressed in hy5 compared to wild type (SupplementalTable S4). ELIP1 was more than 2-fold differentiallyexpressed in hy5 compared to wild type under the con-trol conditions (Supplemental Table S4), and HY5 haspreviously been shown to be involved in the inductionof ELIP1 and establishment of PSII activity during adark-to-light transition (Harari-Steinberg et al., 2001).

The genes that showed a change in expression in thehy5 and cry1 mutants under control conditions wereexcluded from our further analysis, and we focusedonly on the genes that changed in wild type after HLtreatment (992 genes) and that were at least 2-folddifferentially expressed after HL treatment in the cry1or hy5 mutants compared to wild type. Only fourgenes were excluded from the HL list due to misreg-ulation under control conditions in the cry1 mutant,and 39 were excluded for the hy5 mutant (Fig. 3A).These restrictions gave 77 genes and 65 genes that weremisregulated in the cry1 and hy5 in response to HL,respectively (Fig. 3A; Supplemental Tables S5 and S6).The overlap between genes misregulated in responseto HL in both cry1 and hy5 was 26 genes (Fig. 3A).

To understand the CRY1 regulon, we analyzed theexpression profile of wild-type seedlings exposed toBL. Using the same cutoffs as for the HL microarrays

Figure 2. Real-time analysis of ELIP1/2 expression at different blue lightintensities. Wild-type and cry1 seedlings were grown for 7 d incontinuous white light at 23�C and shifted to different BL intensities.Expression of ELIP1 (At3g22840; black bars) and ELIP2 (At4g14690;gray bars) was analyzed using real-time PCR. The fold induction afterBL exposure related to the control of each genotype is presented. Theresults were normalized to the expression level of At4g36800 encodinga ubiquitin-protein ligase-like protein. The mean 6 SE of at least fourbiological replicates is shown.

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(Supplemental Table S1), 858 genes changed 2-fold ormore after exposure to BL in wild type (Fig. 3B). Whenwe compared the expression profiles of wild-type plantsexposed to HL and BL, the overlap was 369 genes; 225genes were 2-fold up-regulated and 144 2-fold down-regulated by both HL and BL (Fig. 3B). Furthermore,the overlap between the genes changing in response toHL and BL in wild type (369 genes) and the misregu-lated genes in cry1 or hy5 was 49 and 38 genes, re-spectively (Table II; Fig. 3B). Expression of 23 of the 26cry1 and hy5 HL-misregulated genes also changed inresponse to high intensity BL in wild type (Table II;Fig. 3B). Thus, it is clear that CRY1 and HY5 are nec-essary for the regulation of a significant group of genesresponding to high irradiance (Table II; Fig. 3). The 49and 38 genes misregulated in cry1 and hy5, respectively,were classified into different categories according to theTAIR gene ontology (http://www.arabidopsis.org/tools/bulk/go/index.jsp; Fig. 3B; Table II). The dominantgroups of genes that were misregulated in the mutantsencode components involved in transcriptional regu-lation, enzymes involved in the phenylpropanoid path-

way, proteins associated with stress responses, andproteins with unknown function (Table II).

Expression of HY5 is induced by HL exposure and theresponse is dependent on CRY1. The genes encoding thetranscription factors PAP1 (PRODUCTION OF AN-THOCYANIN PIGMENT1; MYB75 and At1g56650)and PAP2 (MYB90 and At1g66390; Borevitz et al.,2000) involved in the regulation of components in theflavonoid biosynthesis were misregulated in cry1 andin cry1 and hy5, respectively. The genes encoding com-ponents catalyzing the entry of metabolites into thephenylpropanoid pathway, such as CHORISMATEMUTASE1 (CM1; At3g29200) and PHE AMMONIA-LYASE1 (PAL1; At2g37040) and PAL2 (At3g53260), werefound to be strongly induced by BL in wild type andmisregulated in the cry1 and hy5 mutants (Table II; Sup-plemental Fig. S1). In the middle section of the path-way, expression of genes encoding 4-COUMARATE:CoA LIGASE3 (4CL3; At1g65060) and CHALCONE-FLAVANONE ISOMERASE (CHI; At3g55120) was inhi-bitedinbothcry1andhy5mutants (Table II;SupplementalFig. S1). In addition, genes encoding the components

Figure 3. Flow chart demonstrating the analysis usedto filter genes that were misregulated in cry1 andhy5 compared to wild type after HL (A) and BL (B)exposure.



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Table II. Genes responding to HL and BL in wild type that are misregulated in the cry1 and hy5 mutants

The genes are grouped in functional categories according to the gene ontology at TAIR (http://www.arabidopsis.org/tools/bulk/go/index.jsp). Genes marked with *

were excluded from the HL gene list due to misregulation also in the control samples compared to wild type.

Transcript ID DescriptionCol

HL_GL

Col

BL_GL

Col

HL_cry1 HL

Col

HL_hy5 HL

GO Biological

Process/Molecular Function

GO Cellular

Component

Transcription

cry1

At5g11260 Basic Leu zipper protein HY5 (HY5) 2.92 3.42 2.19 2.36* Transcription factor activity Nucleus

At1g56650 Myb family transcription factor

(MYB75) PAP1

13.31 6.55 2.01 2.55* Transcription factor activity Nucleus

At5g49330 Myb family transcription factor 2.14 4.57 2.12 1.48 Transcription factor activity Nucleus

hy5

At4g09820 Basic helix-loop-helix family protein 5.27 4.62 1.69 4.13 Involved in flavonoid

biosynthesis?

Nucleus

At1g06180 Myb family transcription factor 4.72 6.19 1.89 2.03 Transcription factor activity Nucleus

At4g26150 Zinc finger (GATA type) family protein 2.79 2.74 1.30 2.54 Transcription factor activity Nucleus

cry1 and hy5

At1g66390 Myb family transcription factor, PAP2 11.88 2.04 4.25 9.25 Transcription factor activity Nucleus

Intracellular signaling

cry1

At4g38690 Putative protein phospholipase C

(EC 3.1.4.3) precursor

0.29 0.22 0.36 0.55 Signal transduction/phospholipase

C activity

–

Biosynthesis of phenylpropanoids

cry1

At2g23 910 Cinnamoyl-CoA reductase, putative 6.45 8.53 4.14 5.33* – –

At3g51240 Naringenin 3-dioxygenase/F3H 8.10 9.04 2.48 5.79* Naringenin 3-dioxygenase activity –

At5g08640 FLS1 2.55 4.03 2.83 5.11* – –

hy5

At3g53260 PAL2 2.02 2.86 1.55 2.83 PAL; ammonia ligase activity Cytoplasm

At3g29200 CM1 2.55 4.01 1.71 2.53 Shikimate pathway/CM1 activity Chloroplast

cry1 and hy5

At2g37040 PAL1 3.13 3.80 2.11 3.0 PAL; ammonia ligase activity Cytoplasm

At3g55120 CHI/chalcone isomerase 3.59 4.50 2.05 2.29 Chalcone isomerase activity Chloroplast

At1g65060 4CL3 3.78 5.44 2.48 5.44 AMP-binding, catalytic activity –

C-compound and carbohydrate metabolism

cry1

At4g19170 9-cis-Epoxycarotenoid dioxygenase,

putative

0.19 0.08 0.17 0.61 – Chloroplast

hy5

At1g48100 Polygalacturonase PG1 5.73 6.30 1.09 0.49 Carbohydrate metabolism/

polygalacturonase activity

Mitochondrion

cry1 and hy5

At4g15480 F-box family protein4, indole-3-acetate

b-glucosyltransferase-like protein

4.39 8.37 2.38 2.14 Metabolism/transferase activity –

Lipid, fatty acid, and isoprenoid metabolism

cry1

At2g15090 Fatty acid elongase, putative 0.35 0.49 0.4 0.92 3-Oxoacyl-(acyl-carrier protein)

synthase activity

Endomembrane

system

At1g78510 Solanesyl diphosphate synthase 3.73 3.84 2.03 1.91 Polyprenyl synthesis;

isoprenoid biosynthesis

Chloroplast

cry1 and hy5

At5g48880 3-Keto-acyl-CoA thiolase 2 3.17 3.70 2.04 3.49 Fatty acid

biosynthesis/catalytic activity

Mitochondrion

Other metabolic processes

hy5

At3g09650 Pentatricopeptide

repeat-containing protein

2.33 2.92 1.74 2.20 mRNA binding/processing? Chloroplast, stroma

cry1 and hy5

At1g06000 UDP-glucoronosyl/UDP-glucosyl

transferase family protein

2.95 6.41 2.15 2.28 Metabolism/transferase activity,

transferring glycosyl groups

–

At5g17780 Hydrolase, a/b fold family protein 2.72 4.55 2.79 3.10 Proteolysis and

peptidolysis/DNA binding

Nucleus

At5g60540 SNO Gln amidotransferase

family protein

2.39 3.36 2.96 3.15 Vit B6 biosynthesis Cytosol

At4g37150 Esterase, putative 4.82 2.94 2.07 2.16 Catalytic activity/hydrolase

activity

Mitochondrion

(Table continues on following page.)

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Table II. (Continued from previous page.)


HL_GL

Col

BL_GL

Col

HL_cry1 HL

Col

HL_hy5 HL

GO Biological


GO Cellular

Component

Photosynthesis

cry1

At3g22840 ELIP1 99.91 52.94 8.82 8.21* Photosynthesis light

harvesting/

chlorophyll A-B

binding family protein

Chloroplast

cry1 and hy5

At4g14690

(also cell

rescue)

ELIP2 88.38 128.90 11.72 6.81 Photosynthesis light

harvesting/

chlorophyll A-B


Chloroplast

At1g76570 Chlorophyll a/b-binding

family protein

4.42 2.93 2.10 2.12 Photosynthesis light

harvesting/

chlorophyll A-B


Chloroplast

Response to biotic or abiotic stimulus/stress

cry1

At1g10370 GST, putative (ERD9) 11.56 9.31 2.07 0.67 Aromatic amino acid

family metabolism/

catalytic activity

Cytoplasm

At4g31820 Phototropic-responsive

NPH3 family protein

0.31 0.38 0.43 0.86 DNA methylation/

DNA binding

–

At5g44680 Methyladenine

glycosylase

family protein

0.15 0.30 0.44 1.13 DNA repair/DNA-

3-methyladenine

glycosylase

I activity

Chloroplast

At1g74670 GA-responsive

protein, putative;

GAST1 like

0.05 0.13 0.18 0.42* – Endomembrane

system

hy5

At2g42530 Cold-regulated protein 14.96 17.59 1.13 2.78 – Cytoplasm

At1g02940 GST, putative 2.48 2.19 1.31 4.08 N-terminal protein

myristoylation/toxin

catabolism

Mitochondrion/

cytoplasm

cry1 and hy5

At4g31870 GPX7 22.85 12.88 6.52 15.46 Glutathione peroxidase

activity

Chloroplast

At2g41000 DNAJ heat shock protein,

putative

2.48 2.02 2.18 3.11 – Endomembrane

system

DNA repair

cry1 and hy5

At5g24850 Cryptochrome dash

(CRYD)

2.38 3.97 2.53 3.28 DNA repair/

deoxyribodipyrimidine

photolyase activity

Chloroplast/

mitochondrion

Assembly of protein

complexes

cry1 and hy5

At5g56090 Cytochrome oxidase

assembly

family protein

2.37 2.0 2.31 2.20 Protein complex

assembly

Chloroplast

Transport

cry1

At5g62210 Embryo-specific

protein related

5.12 7.10 2.91 5.05* Transport /receptor

activity

Endomembrane

system

At5g02270 ABC transporter-like

protein

NBD-like protein POP

2.31 5.46 2.30 4.36* Transport/ATP-binding

cassette (ABC)

transporter activity

Membrane

At1g52190 Proton-dependent

oligopeptide

transport family protein

0.16 0.36 0.21 0.78 Transporter activity Membrane

At5g48490 Protease inhibitor/seed

storage/lipid transfer

protein family protein

0.14 0.33 0.26 1.15 Lipid transport/

lipid binding

Endomembrane

system

(Table continues on following page.)



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catalyzing the final steps, F3H (At3g51240) and FLS(At5g08640), are regulated via CRY1 in response to HL(Table II; Supplemental Fig. S1).

Stress-related genes, such as the putative GLUTA-THIONE PEROXIDASE7 (GPX7) gene (At4g31870),were strongly up-regulated in wild type in responseto HL and BL, and the induction of GPX7 was sup-pressed in cry1 and hy5. Furthermore, the expressionof At1g10370 and At1g02940, both encoding glutathi-one S-transferases (GSTs), was misregulated in cry1and hy5, respectively (Table II). Genes encoding fur-ther potential components in stress response such asubiquinone methyltransferase (At2g41040) and PYR-IDOXIN BIOSYNTHESIS2 (PDX2; At5g60540) werealso misregulated in response to HL in the cry1 mutant(Table II). The microarray analysis also confirmed thesuppressed induction of ELIP1/2 in the cry1 and hy5

mutants shown with real-time PCR. However, ELIP1 isnot grouped as misregulated in hy5 (Table II) because itwas already more than 2-fold differentially expressedin hy5 under the control growth conditions.

CRY1 and HY5 Regulate HL-Responsive Genes viaG-Box Promoter Elements

HY5 has previously been demonstrated to bind aG-box motif (Gao et al., 2004). We examined the 500-bppromoter sequences of all cry1 and/or hy5 HL-misregulated genes (116 in total [77 1 65]) to determinethe frequency of occurrence of the G-box consensus(Fig. 3A). Within the promoters of the 26 genes mis-regulated in both cry1 HL and hy5 HL (Fig. 3A), a classicG box (CACGTG) was found in five promoters (19%;Table III). The G-box frequency in the hy5-specific HL

Table II. (Continued from previous page.)


HL_GL

Col

BL_GL

Col

HL_cry1 HL

Col

HL_hy5 HL

GO Biological


GO Cellular

Component

hy5

At4g01660 ABC1 family protein,

putative

ABC transporter

2.49 2.54 1.85 2.40 – –

At1g06690 Aldo/keto reductase

family protein

2.09 2.88 1.96 2.98 Transport Cytoplasm

Developmental processes

cry1

At2g40610 Expansin, putative (EXP8) 0.05 0.23 0.19 0.33* Cell wall

modification

Extracellular

Biological process unknown

cry1

At2g34620 Mitochondrial transcription

termination factor related

0.25 0.17 0.48 0.56 – Chloroplast

At1g25230 Purple acid phosphatase

family protein

0.12 0.18 0.45 0.53 Phosphatase/

hydrolase activity

Endomembrane

system

hy5

At5g35970 DNA-binding protein, putative;

DNA helicase like

2.50 4.16 1.76 2.65 DNA binding Chloroplast

cry1 and hy5

At1g23010 Multicopper oxidase type I

family protein

3.54 3.50 2.68 5.21 Cell-matrix adhesion/copper

ion binding

Cytoskeleton

At5g52250 Transducin family protein/WD-40 repeat

family protein, contains

similarity to COP1

3.33 3.87 2.99 4.18 – Chloroplast

At2g25530 AFG1-like ATPase family protein 2.88 4.60 2.55 2.49 ATP binding –

At2g41040 Methyltransferase related 2.90 3.04 2.36 2.3 Methyltransferase activity Chloroplast/plastoglobule

(Vidi et al., 2006;

Ytterberg et al., 2006)

At1g80440 Kelch repeat-containing F-box

family protein

0.11 0.14 0.28 0.31 – –

Unknown proteins

cry1

At3g17610 Unknown protein 3.69 4.78 2.17 1.58 – –

At3g02170 Unknown protein 0.39 0.40 0.50 0.99 – –

At5g50335 Expressed protein 0.35 0.12 0.39 0.71 – Endomembrane system

At5g16030 Putative protein 0.13 0.45 0.44 0.71 – –

hy5

At1g16850 Unknown protein 16.98 9.15 1.48 3.41 – Endomembrane system

At2g28400 Unknown protein 16.03 3.59 1.39 0.46 – Mitochondrion

At4g36530 Putative protein 2.54 2.34 1.64 2.56 Proteolysis and

peptidolysis/catalytic activity

Cytoplasm

cry1 and hy5

At3g44450 Expressed protein 2.13 3.97 2.69 2.16 – –

At2g18300 Unknown protein 0.10 0.28 0.33 0.46 Regulation of transcription –

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regulon was 23% (9/39 genes; Table III). These num-bers represented a significant enrichment versus thegenomic average for classic G-box elements withinArabidopsis 500-bp promoter spaces (10.3% in theTAIR annotated Arabidopsis genome, v.6). Inclusion ofG-box sequence variants increased these frequenciesto 35% (14/26 cry1 1 hy5 HL overlap) and 56% (22/39hy5-specific HL genes), respectively (Table III). Fur-thermore, in the 51 genes specific to the cry1 HLregulon (Fig. 3C), 16 (31%) promoters contained aclassic G box (CACGTG), and addition of the G-boxvariants (CACGTH) collectively accounted for another24% (12/51) of the cry1-specific HL gene set. Thus, theCACGTH frequency was 55% in cry1-specific HL generegulon (Table III).

The CRY1 and HY5 regulons identified in our arrayexperiments using stringent statistical selection crite-ria (logit-t P , 0.025 and uniform 2-fold change inexpression) were small and therefore prohibitive forfurther bioinformatic analyses such as in silico muta-genesis and novel cis-element detection. Subsequentcis-regulon analyses of our microarray data thereforeused sliding-scale fold-change standards in combina-tion with a novel motif bioactivity-testing protocol(Benedict et al., 2006; Geisler et al., 2006). The algo-rithm used reduces false positives by correlating thenaturally occurring distribution of cis-elements in the

Arabidopsis genome with the specific HL or cry1- andhy5-responsive genes (Geisler et al., 2006). Using thenew fold-change criteria, the total size of the wild-typeHL differentially expressed gene regulon increased to1,577 genes (820 induced and 757 repressed), the wild-type BL regulon increased to 1,516 genes (634 inducedand 882 repressed), the cry1 HL-misregulated generegulon increased to 371 genes (217 induced and 154repressed compared to wild type), and the hy5 HL-misregulated gene regulon increased to include 707genes (348 induced and 359 repressed compared towild type). Genes containing the classic G box withintheir 500-bp promoter demonstrated a strong overrep-resentation in the wild-type HL- and BL-inducedregulons (Table III). The G box was also overrepre-sented in the regulons of genes repressed in cry1 andhy5 HL-treated seedlings compared to wild type. Byperforming in silico mutagenesis (Benedict et al., 2006;Geisler et al., 2006) of the classic G-box sequence inwild-type HL, hy5 HL, and cry1 HL backgrounds, wewere able to establish that the palindromic G-box con-sensus could vary at position 1 (or inversely, position 6)without loss of bioactivity (Supplemental Fig. S3).

A more general survey of cis-elements previouslyreported to contribute to light-, phytochrome-, crypto-chrome-, and HY5-regulated transcription (PLACE data-base; http://www.dna.affrc.go.jp/PLACE; Gao et al.,

Table III. Known and novel cis-regulons demonstrating significant (x2, P , 0.05) transcriptional responses during exposure HLand BL in wild-type, cry1, and hy5 Arabidopsis backgrounds

x2 expected values were calculated based on the whole-array response frequencies. ind, Induced genes; rep, repressed genes.

cis-Element

Name

cis-Element

ConsensusAnnotation

Gene Regulons Demonstrating

cis-Regulon Over-/Underrepresentation

x2 P Value

(Enriched/Reduced)

G box CACGTG Light and phyA response wtHL/wtGL ind 3.5 E-49 (en)wtBL/wtGL ind 4.6 E-15 (en)cry1HL/wtHL rep 3.6 E-03 (en)hy5HL/wtHL rep 2.3 E-02 (en)

G-box variants CACGTH Light and phyA response wtHL/wtGL ind 9.8 E-30 (en)wtBL/wtGL ind 5.6 E-09 (en)wtBL/wtGL rep 2.5 E-02 (en)cry1HL/wtHL rep 1.5 E-05 (en)hy5HL/wtHL rep 1.2 E-02 (en)

I box GATAAGR Phy, Cry, and plastid response wtHL/wtGL ind 2.2 E-02 (red)wtHL/wtGL rep 2.0 E-12 (en)wtBL/wtGL ind 8.6 E-04 (red)wtBL/wtGL rep 3.0 E-13 (en)cry1HL/wtHL ind 1.1 E-03 (en)hy5HL/wtHL ind 1.3 E-02 (en)

MYC/MYB site CATGTG Binds MYC and MYB TFs wtHL/wtGL rep 6.4 E-09 (en)wtBL/wtGL rep 1.5 E-02 (en)cry1HL/wtHL ind 1.7 E-02 (en)hy5HL/wtHL ind 1.7 E-03 (en)

CryR1 GnTCKAG Cry-associated HL response wtHL/wtGL ind 6.6 E-03 (en)cry1HL/wtHL rep 2.4 E-02 (en)

CryR2 ACATAwCT Cry-associated HL response wtHL/wtGL rep 1.2 E-02 (en)wtBL/wtGL rep 3.6 E-02 (en)cry1HL/wtHL ind 1.9 E-03 (en)cry1HL/wtHL rep 4.1 E-02 (en)

HycR1 ACmyACAy HY5- and Cry-associated HL response wtHL/wtGL rep 7.4 E-04 (en)cry1HL/wtHL ind 2.0 E-03 (en)hy5HL/wtHL ind 2.9 E-03 (en)



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2004) showed that the promoters of genes differen-tially regulated during BL and HL treatment of wildtype, and HL treatment of cry1 and hy5 seedlings,were also enriched for the I-box and myelocytomatosisoncogene (MYC) cis-elements. The respective cis-regulons were significantly more likely to be inducedor repressed in response to HL than the general pop-ulation of genes on the microarray (Table III). In anattempt to identify novel cis-elements, we used theGibbs sampling-based Inclusive Motif Sampler pro-gram (Thijs et al., 2002) to identify 8- to 10-bp con-sensus sequences overrepresented in the 500-bppromoters of the genes associated with the CRY1-and HY5-mediated transcriptional responses to HL(Supplemental Tables S5 and S6). Excluding hits re-sembling the G box, the most significant cry1 HL-repressed, regulon-enriched element was GnTCKAG(CryR1; Table III). Bioactivity of CryR1 was indicatedby the significant overrepresentation in the pro-moters of genes induced by HL in wild type. Anotherelement identified in the list of cry1 HL-inducedgenes was ACATAwCT (CryR2; Table III). Bioactivityof the CryR2 element was also indicated by a sig-nificant repression at frequencies greater than thatpredicted by random chance of the genes where thepromoter contains the element following HL and BLtreatments in wild type. In addition, in silico muta-genesis of the CryR1 and CryR2 elements demon-strated that the CryR1 element could not varywithout losing biological activity, whereas theCryR2 element could vary only at position 2 withoutloss of bioactivity (Supplemental Fig. S4). Thus,genes containing the CryR1 (GnTCKAG) and thetwo CryR2 variants (ACATAwCT and ADATAwCT)were all significantly induced and repressed, respec-tively, in response to HL in wild type (SupplementalFig. S4). Furthermore, genes containing these ele-ments were repressed or induced in the cry1 mutantcompared to wild type (Supplemental Fig. S4). An-other element, HycR1 (ACmyACAy), was identifiedfrom the cry1 HL-induced gene regulon, and bioac-tivity was confirmed in cry1 and hy5 HL-treatedplants (with significant cis-regulon enrichment in theinduced gene groups for these mutants; Table III).

The cry1 and hy5 Mutants Demonstrate a DefectiveStress Response

In wild type, anthocyanin accumulation increasedwith prolonged exposure to high irradiance, and after24-h exposure, more than a 3-fold increase was ob-served (Supplemental Fig. S2). In contrast, anthocya-nin contents were unchanged in the cry1 and hy5mutants after 9-h exposure to HL (Supplemental Fig.S2), supporting the misregulation of genes encod-ing components of the phenylpropanoid pathway ob-served in cry1 and hy5 (Supplemental Fig. S1).Reduced anthocyanin levels in CRY1-deficient Arabi-dopsis seedlings have been shown in continuous bluelight (Ahmad et al., 1995; Lin et al., 1996) and in con-

tinuous white light (Neff and Chory, 1998). However,our results demonstrate that CRY1 and HY5 also playa key role in the high irradiance-induced accumulationof anthocyanin. HY5 is subject to regulation via COP9signalosome-mediated degradation in the dark(Hardtke et al., 2000; Osterlund et al., 2000). To verifythat HY5 protein itself was stable under HL condi-tions, western blots were performed, and no reductionin HY5 protein could be detected in HL samples com-pared to LL samples (Supplemental Fig. S5).

Figure 4. Representative photographs of 5-week-old plants of the cry1mutant (A) and wild type (B) following 24-h exposure to HL. Photo-graphs of 7-d-old seedlings of the cry1 mutant (C) and wild type (D andE) following 24-h exposure to HL. F, Chlorophyll content in control-grown and 24-h HL-exposed seedlings of the cry1 mutant and wildtype. The mean 6 SD of four biological replicates is shown. One-wayANOVA followed by Bonferroni’s multiple comparison test to comparecolumn pairs (wild-type control versus wild-type HL and cry1 controlversus cry1 HL) was performed using GraphPad Prism version 4.00 forWindows (GraphPad Software, www.graphpad.com). ***, P , 0.001.

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Exposure to HL resulted in a gradual photoinacti-vation of PSII as demonstrated by a drop in Fv/Fmfrom 0.83 to 0.73 after only 3 h exposure and furtherdown to 0.59 after 12 h in wild type (Table I). Despitethe reduced accumulation of anthocyanin in the hy5mutant, the sensitivity to HL exposure was similar towild type in the mutant (Table I). In contrast, the cry1mutant was twice as sensitive to the HL exposure aswild type, shown by a drop in Fv/Fm from 0.83 to 0.32(Table I). Furthermore, photo bleaching after pro-longed (24 h) exposure to HL was clear in the cry1 mu-tant compared to wild type and the hy5 mutant, bothin seedlings and in 5-week-old plants (Fig. 4). The HL-sensitive phenotype of the cry1 mutant supports theconclusion from the CRY1 regulon that a significantcomponent of the HL photoprotective response ismediated by CRY1.

DISCUSSION

The role of cryptochromes during early light re-sponse (hours of light exposure) has been well estab-lished, and expression profiles of the Arabidopsis cry1mutants revealed that a large number of genes areregulated via cryptochromes (Ma et al., 2001; Foltaet al., 2003; Jiao et al., 2003; Ohgishi et al., 2004).However, in 7-d-old seedlings grown at 100 mmolquanta m22 s21, only 48 genes were differentiallyregulated in the cry1 mutant compared to wild type(Supplemental Table S3). Exposure of the cry1 mutantto HL revealed that 77 HL-responsive genes weredifferentially expressed in the mutant compared towild type exposed to HL (Fig. 3A). Furthermore, 49 ofthe 77 HL genes misregulated in the cry1 mutant werealso induced in wild type by high intensity BL (Fig.3B). The remaining 28 CRY1-dependent genes requirelight qualities outside the narrow waveband used inthese BL experiments, possibly through functionalinteraction between cryptochrome and phytochromes(Ahmad et al., 1998b; Mas et al., 2000). Thus, a largenumber of genes specific to the high light responsewere discovered to be mediated via the photoreceptorCRY1, and we have described a novel function ofCRY1 as a mediator of plant responses to high irradi-ance controlling the expression of a large number ofHL-responsive genes.

During exposure to high irradiance, CRY1 promotesthe expression of genes associated with stress protec-tion mechanisms such as GPX7, encoding a putativeglutathione peroxidase (At4g31870) and the GSTERD9 (At1g10370; Table II). The GST proteins havebeen shown to respond to various stresses such as HL,cold, and drought (Wagner et al., 2002; Seki et al., 2003;Goulas et al., 2006). After exposure to HL, six differentGST genes were induced, and four of them wereinduced in both HL and high intensity BL (Supple-mental Tables S1 and S2). Thus, our results suggestthat GSTs play an important role in the response tohigh light stress. In addition, genes encoding a poten-

tial ubiquinone methyltransferase (At2g41040) andPDX2 (At5g60540) are misregulated in response toHL in the cry1 mutant (Table II). The gene At2g41040has a predicted ubiquinone methyltransferase domainand the encoded protein was recently reported to befound in plastoglobuli (Vidi et al., 2006; Ytterberg et al.,2006) and could potentially be involved in phylloqui-none (vitamin K1) biosynthesis. Phylloquinones serveas electron acceptors in the PSI reaction center and arecritical for photosynthetic function. PDX2 togetherwith PDX1 form a Gln amidotransferase complex in-volved in vitamin B6 biosynthesis (Tambasco-Studartet al., 2005). Vitamin B6 has been shown to be a potentantioxidant with the ability to quench ROS such assinglet oxygen and superoxide in human erythrocytes(Jain and Lim, 2001) and fungus (Ehrenshaft et al.,1999) and prevents lipid peroxidation in human eryth-rocytes (Jain and Lim, 2001). Presumably, vitamin B6could also protect photosynthetic membranes againstlipid peroxidation caused by ROS production duringhigh light stress. Taken together, these data indicate animportant role of CRY1 in modulating the response ofplants to changes in irradiance leading to oxidativedamage.

Blue light receptors have been described to regulatea range of different plant responses, including deetio-lation, photo entrainment of the circadian clock, pho-totropic curvature, and chloroplast relocation. Inaddition to CRY1, three different blue light receptorswith known functions have been described in Arabi-dopsis, CRY2, and the phototropins PHOT1 andPHOT2. The cry2 mutant did not show impaired ex-pression of our HL marker genes after exposure tohigh irradiance (Fig. 1). The large number of genes thatwere misregulated in response to HL in the cry1 mu-tant excludes redundant roles of CRY1 and CRY2 un-der the HL conditions. The underlying reason for thismay be found in the different behavior of the twoproteins; the CRY2 protein is rapidly degraded byUV-A, blue, and green light, whereas the CRY1 proteinis stable (Ahmad et al., 1998a; Lin et al., 1998). CRY2 isparticularly important in retarding hypocotyl growthin response to low intensity blue light, whereas CRY1has a prevalent role in response to higher blue lightintensity (Lin et al., 1998). Furthermore, CRY1 phos-phorylation kinetics and activation exhibits a positivecorrelation with blue light intensity in direct contrastto CRY2, which exhibits a negative correlation withblue light intensity (Shalitin et al., 2002, 2003). Thus,although CRY1, but not CRY2, has been shown tomediate a response to higher irradiances, a role forCRY1 during changes in irradiance and the high lightresponse has not been previously described. Related tothis proposed role of CRY1 in mediating light stressresponse, the phototropin PHOT2 was shown to reg-ulate the avoidance movement of chloroplasts in re-sponse to high intensity blue light (Jarillo et al., 2001;Kagawa et al., 2001; Sakai et al., 2001). In low light,chloroplasts are distributed along the periclinal wallsin a manner that presumably maximizes light capture



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for photosynthesis, whereas under high fluence rates,chloroplast damage is minimized by movement of thechloroplasts toward the anticlinal walls (Jarillo et al.,2001). This raises the possibility that the CRY1 andPHOT2 responses share similar mechanisms. How-ever, earlier studies have shown that the cry1cry2double mutant shows normal blue light-induced chlo-roplast relocation (Kagawa and Wada, 2000), indicat-ing that these two classes of photoreceptors, CRY1 andPHOT2, act through separate pathways to provideprotection against excess light.

Twenty-six genes of the 77 CRY1-dependent geneswere also misregulated in response to HL in the hy5mutant, and 23 of those 26 genes are also regulated byBL in wild type (Fig. 3, A and B). HY5 is transcrip-tionally activated in a phytochrome-dependent man-ner in etiolated seedlings exposed to light (Teppermanet al., 2001). Interestingly, expression of HY5 and theamount of HY5 protein was induced by HL exposure(Table II; Supplemental Fig. S5), and the response toHL was dependent on CRY1 activity (Table II). Theexpression profiles of cry1 and hy5 demonstrated thatCRY1 and HY5 are connected in the response to HLbut that they also play distinct roles (Fig. 5). Genesencoding enzymes associated with the phenylpropa-noid pathway are represented in the group of genesmisregulated in both cry1 and hy5 mutants (Table II).Expression of genes encoding the CHS has previouslybeen demonstrated to be regulated by blue light ina CRY-dependent manner (Fuglevand et al., 1996).However, we demonstrate here that genes encodingcomponents at every level of the phenylpropanoidpathway, from transcriptional control to the final en-zymatic steps of the pathway, are regulated by a CRY1-and HY5-mediated BL response (Supplemental Fig.S1). After 12-h exposure to HL, no anthocyanin accu-mulation could be detected in the hy5 seedlings (Sup-plemental Fig. S2). Furthermore, the induction of

ELIP1/2 and UGT84A1 was impaired in the cry1 andhy5 mutants following exposure to HL. It has previ-ously been demonstrated that the loss of HY5 impairsthe light-induced expression of ELIP1 (Harari-Steinberget al., 2001) and the UV-B-responsive expression ofseveral genes such as ELIP1/2 and UGT84A1 (Ulmet al., 2004; Brown et al., 2005). In addition to the effecton gene expression following exposure to UV-B, hy5demonstrated a UV-B sensitive phenotype, and HY5was found to be a key component of the UVR8pathway required for survival under UV-B radiation(Brown et al., 2005; Oravecz et al., 2006). The overlapbetween the genes misregulated in the hy5 mutantcompared to wild type, in response to UV-B, and tohigh intensity blue light was small (only five genes),indicating that the UV-B and HL responses are trig-gered by two separate mechanisms. Thus, our resultsdemonstrate that in addition to a role in photomor-phogenesis and in the UV-B response, HY5 is a keycomponent of the CRY1-mediated pathway in re-sponse to HL. In addition, 39 genes were misregulatedin the hy5 mutant in response to HL in a CRY1-independent manner (Fig. 5). Two genes encodingcomponents related to pathogen defense signalingELI3-2 (At4g37990) and a WRKY family transcriptionfactor, WRKY70 (At3g56400; Kiedrowski et al., 1992; Liet al., 2004), are misregulated in the hy5 mutant (Sup-plemental Table S6), suggesting that HY5 may be a keycomponent of the cross talk between light and path-ogen defense signaling. HY5 has been demonstrated tobind a G-box motif (Gao et al., 2004), and, consistentwith this, bioinformatic analysis of our array datarevealed that a classic G-box (CACGTG) or a G-boxvariant (CACGTH) were significantly enriched in theHY5 regulon versus the genomic average for theseelements within Arabidopsis 500-bp promoter spaces(Table III).

A large number of genes encoding componentsnecessary for the photoprotective response in plantsare misregulated in the cry1 mutant. The effect of thislimited response was shown in the cry1 mutant withthe 60% loss of Fv/Fm after a 12-h high light exposurecompared to around 30% loss in hy5 and wild type(Table I). Furthermore, photo bleaching after pro-longed (24 h) exposure to high light was exacerbatedin the cry1 mutant compared to wild type both inseedlings and in 5-week-old plants (Fig. 4). Thus, thecry1 mutant has more difficulty recovering from highlight exposure than wild type. However, the cry1 mu-tant was reported to be able to acclimate photosyntheticcapacity to high light after long-term (weeks) expo-sure (400–600 mmol quanta m22 s21; Walters et al., 1999;Weston et al., 2000). However, Weston et al. (2000)demonstrated that a shift directly to 600 mmol quantam22 s21 without a pretreatment with 400 mmol m22 s21

resulted in a severe light stress in the cry1 mutantcompared to wild type. Even though a direct compar-ison with the results of Weston et al. (2000) cannot bemade, our results confirm an essential role for CRY1during high light stress response. Despite the largeFigure 5. Working model for the CRY1-mediated HL response.

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number of genes, including ELIP1/2 and componentsof the phenylpropanoid pathway found to be misre-gulated in both hy5 HL and cry1 HL, the tolerance tophotoinhibition was not changed in hy5 (Table I). Adouble null ELIP Arabidopsis mutant did not demon-strate reduced tolerance to photoinhibition and pho-tooxidative stress compared to wild type, indicatingthat the ELIP proteins do not have a photoprotectivefunction (Rossini et al., 2006). The difference in lightstress sensitivity between cry1 and hy5 mutants may beexplained by the fact that CRY1 is the mediator of thehigh irradiance response and thereby controls a largernumber of genes compared to the downstream com-ponent HY5 (Fig. 4). At this point, we can onlyspeculate about which of the CRY1-specific genesencode key components of the protective mechanismsagainst light stress. However, it is worth noting thatthe MYB family transcription factor (At5g49330),At3g17610, encoding a protein with unknown functionand the GSTs (ERD9) are all strongly induced in wildtype by HL and BL and misregulated specifically incry1.

Light-regulated protein degradation is central tocryptochrome signaling, and CRY1 was found to inter-act with the E3 ubiquitin ligase CONSTITUTIVELYPHOTOMORPHOGENIC1 (COP1; Yang et al., 2001).COP1 is required for light-regulated degradation of sev-eral transcription factors involved in light-regulatedtranscription, including HY5. It was proposed that alight-driven conformational change of the cryptochromesinduces a structural modification of COP1 that releasesHY5 bound by COP1 in the dark (Cashmore, 2003).Furthermore, a crucial role for COP1 as a positive reg-ulator of the UV-B response was recently demon-strated (Oravecz et al., 2006). It is possible that COP1is involved in the CRY1-mediated high irradiance re-sponse we have reported here, and future work willdemonstrate if the CRY1-COP1-HY5 signaling systemthat is used to regulate photomorphogenesis alsoplays a role in the response to high irradiances. Twonovel cis-elements were enriched in the list of CRY1-dependent HL-responsive genes, CryR1 (GnTCKAG)and CryR2 (ACATAwCT; Table III). CryR1 was signif-icantly enriched in the promoters of genes induced byHL in wild type, suggesting interaction with an acti-vator of gene expression. In contrast, the CryR2 ele-ment was significantly enriched in the promoters ofgenes repressed by HL in wild type, suggesting inter-action with a repressor of gene expression (Table III).Thus, we have identified two novel potential CRY1-associated HL response elements, CryR1 and CryR2(Fig. 5). Future work will reveal whether these ele-ments are targets of the CRY1-mediated high irradi-ance transcriptional response.

CONCLUSION

Analysis of the high irradiance response of the pho-toreceptor mutants phyA, phyB, cry1, and cry2 and thetranscription factor mutants hy5 and hyh revealed a

novel function of CRY1 in mediating plant responsesto high irradiances. In addition to a role in photomor-phogenesis, CRY1 is essential to the induction ofphotoprotective mechanisms against high light stress.Thus, we have demonstrated that high irradiance sig-nals can be transduced in a chloroplast-independentmanner by cytosolic/nucleic components.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Seeds from Arabidopsis (Arabidopsis thaliana) Columbia-0 (Col-0) and

phyA-211, phyB-9, cry1-304 (Ahmad and Cashmore, 1993), cry2-1 (Guo et al.,

1998), and hy5 (Maxwell et al., 2003) and hyh (WiscDsLox253D10) were

obtained from TAIR. Arabidopsis seeds were sterilized (75% [v/v] ethanol,

0.01% [v/v] Triton X-100) for 15 min and washed three times with 95% (v/v)

ethanol before spreading onto 0.27% (w/v) phytoagar plates containing

13 Murashige and Skoog basal salt mixture including vitamins (Duchefa) and2% Suc. The plates were stratified 2 d in darkness at 4�C and then placed either7 d into continuous white light (100 mmol quanta m22 s21, 23�C or weretransferred to soil after 10 d. For HL treatment, seedlings or 4- to 5-week-old

plants were transferred for 3 or 12 h to 1,000 mmol quanta m22 s21 (metal

halide HQI-T 400 W daylight light bulbs, Osram). For BL exposure, the HQI-T

400 W lamps were filtered through color filter number 74, 400 to 540 nm with

an absorption maximum of 470 nm (Night Blue; Rosco International). Air

temperature was 22�C. Seedlings (at least 10) were harvested and directlyfrozen in liquid nitrogen. All experiments were performed using at least three

biological replicates.

Analysis of Anthocyanin Content

Relative anthocyanin levels were determined according to Neff and Chory

(1998). In brief, 50 to 70 mg seedlings were incubated overnight in 450 mL

methanol acidified with 1% HCl. After the addition of 250 mL distilled water,

anthocyanins were separated from chlorophylls with 625 mL chloroform. The

anthocyanin content was determined by measuring A530 and A657 of the

aqueous phase and subtracting 0.25 3 A657 from the A530 value.

PSII Photochemistry

In vivo chlorophyll fluorescence was measured using a modulation

fluorometer PAM 101-103 (Heinz Walz) from the adaxial side of excised leaf

material. The nomenclature of van Kooten and Snel (1990) was used for the

parameters of chlorophyll fluorescence. The maximal photochemical effi-

ciency of PSII photochemistry in the dark-acclimated state was evaluated as

Fv/Fm 5 (Fm 2 Fo)/Fm (van Kooten and Snel, 1990) after 1 h acclimation todarkness. In both the light- and dark-acclimated states, the minimal fluores-

cence intensity was measured by analytic modulated light, the maximal

fluorescence intensity by saturating pulses (flash light intensity approximately

4,000 mmol photons m22 s21) of 0.8 s duration.

RNA Isolation

For total RNA isolation, the RNeasy Plant Mini kit (Qiagen) was used

according to the manufacturer’s instructions. The concentration of total RNA

was determined with a Nanodrop ND-1000 spectrophotometer.

cDNA Synthesis and Real-Time PCR

cDNA was prepared from 1 mg of total RNA by using the iScript cDNA

Synthesis kit (Bio-Rad) according to the manufacturer’s instructions. cDNA

was diluted 10-fold, and 2 mL of the diluted cDNAwas used in a 20-mL iQ SYBR

Green Supermix reaction (Bio-Rad). All reactions were performed in triplicate.

The following primers were used: ELIP1 (At3g22840) forward primer, 5#-CGT-TGCCGAAGTCACCAT-3#, reverse primer, 5#-AATCCAACCATCGCTAAA-CG-3#; ELIP2 (At4g14690) forward primer, 5#-CACCACAAATGCCACAGTCT-3#,reverse primer, 5#-TGCTAGTCTCCCGTTGATCC-3#; LHCB2.4 (At3g27690)



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forward primer, 5#-GCCATCCAACGATCTCCTC-3#, reverse primer, 5#-TGG-TCCGTACCAGATGCTC-3#; cytosolic APX2 (At3g09640) forward primer,5#-CAAGGAGCTGTTCCCTATTCTG-3#, reverse primer, 5#-GAGGTGGCT-CAACTTTGTCC-3#; and ubiquitin-protein ligase-like protein (At4g36800)forward primer, 5#-CTGTTCACGGAACCCAATTC-3#, reverse primer, 5#-GGA-AAAAGGTCTGACCGACA-3#. The primers were designed to flank intronsites to make it possible to detect amplification of genomic DNA. Thermal

cycling consisted of an initial step at 95�C for 3 min, followed by 40 cycles of 10 sat 95�C, 30 s at 55�C, and 10 s at 72�C, after which a melting curve wasperformed. Real-time PCR was monitored by using the MyiQ Single Color

Real-Time PCR Detection system (Bio-Rad). The adjustment of baseline and

threshold was done according to the manufacturer’s instructions. The relative

abundance of ELIP1, ELIP2, APX2, and LHCB2.4 transcripts was normalized to

the constitutive expression level of ubiquitin-protein ligase-like protein

mRNA. The data were analyzed by using LinRegPCR (Ramakers et al., 2003)

and according to Pfaffl (2001).

Microarray Analysis

cRNA Synthesis and Hybridization toAffymetrix GeneChips

RNA quality was assessed by agarose gel electrophoresis and spectropho-

tometry. RNA was processed for use on Affymetrix Arabidopsis ATH1

GeneChip arrays, according to the manufacturer’s protocol. Five micrograms

of total RNA of each of the different pools of Col-0, cry1-4, and hy5-1 seedlings,

treated with high light for 0 or 3 h and Col-0 subjected to 3 h high light plus a

blue light filter (filter no. 74, Night Blue, 400–540 nm, absorption maximum of

470 nm; Rosco International) was processed and hybridized to a Genechip

Arabidopsis ATH1 Genome Array according to the manufacturer’s instruc-

tions (Affymetrix). In brief, 5 mg of total RNA was used in a reverse tran-

scription reaction (Ambion MessageAmp kit) to generate first-strand cDNA.

After second-strand synthesis, double-stranded cDNA was used in an in vitro

transcription reaction to generate biotinylated cRNA. The quality of purified

and fragmented cRNA was assessed by spectrophotometry and agarose gel

electrophoresis. A total of 15 mg of fragmented, biotinylated cRNA was used

for hybridization. Hybridization, washing, staining, and scanning procedures

were performed as described in the Affymetrix technical manual. A Hybrid-

ization Oven 640, a Fluidics Station 450, and a GeneChip Scanner 3000 were

used. MIAME information describing the samples, as well as raw microarray

data, including Affymetrix.CEL files, have been deposited in the National

Center for Biotechnology Information Gene Expression Omnibus (GEO;

http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series

accession number GSE7743.

Data Analysis

Normalization and expression estimate computation were calculated from

the .CEL output files from the Affymetrix GCOS 1.1 software using gcRMA

implemented in R using standard settings. Statistical testing for differential

expression was performed with logit-t analysis (P , 0.025; [21]). The .CHP andlogit-t files were loaded into GeneSpring 7.3 (Agilent Technologies). Affyme-

trix present, marginal, and absent flags were used as an indicator of whether

or not a gene was expressed. Genes called absent in both of the compared

conditions were removed from subsequent analyses.

Bioinformatic Analysis

Analyses of the Affymetrix ATH1 microarray data to determine cis-

regulon activity, in silico mutagenesis result, and novel cis-element enrich-

ment were performed as previously described (Benedict et al., 2006; Geisler

et al., 2006). Briefly, the normalized microarray data (reported as fold-change

values for each comparison) were entered into a spreadsheet program so that

the expression of each gene (in rows) could quickly be read across to find

induction/suppression fold-change values for all treatments (in columns).

Fold-change data were then numerically discretized for each gene on the array

into the categories of not present (2), nonresponsive (0), induced (11), orrepressed (21), using the sliding-scale fold-change standard described inBenedict et al. (2006). By downloading the list of all genes containing a cis-

element of interest from TAIR (http://www.arabidopsis.org/) and filtering

the whole array gene dataset/spreadsheet for only these genes, x2 compar-

isons in the induction and repression frequencies for cis-regulons versus the

array population as a whole could be performed to assess bioactivity. Novel

cis-elements identified as enriched in the gene lists reported in Supplemental

Tables S1 to S6 using the Inclusive Motif sampler program (Thijs et al., 2002;

http://homes.esat.kuleuven.be/;thijs/Work/MotifSampler.html) were alsotested using the x2 comparison of cis-regulon induction/repression frequency

versus general array population induction/repression frequency.

Protein Extraction, Western Blot, andChlorophyll Extraction

Proteins were extracted according to Hurry et al. (2000) and the SDS-PAGE

according to Lundmark et al. (2006). The HY5 antibody was provided by Santa

Cruz Biotechnology. The chlorophyll was extracted and analyzed according to

Porra et al. (1989).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Schematic overview of the expression of genes

encoding enzymes in the flavonoid pathway in Arabidopsis in re-

sponse to HL in wild type, cry1, and hy5 and to high intensity BL in

wild type (based on the KEGG pathway).

Supplemental Figure S2. Quantification of anthocyanin content in wild

type, cry1, and hy5 seedlings grown in GL conditions at 23�C andshifted to HL for the time period indicated.

Supplemental Figure S3. In silico mutagenesis of the palindromic G-box

binding consensus (wild type 5 CACGTG).

Supplemental Figure S4. In silico mutagenesis of the CryR1 (GnTCKAG)

and CryR2 (ACATAwCT) consensus sequences.

Supplemental Figure S5. Anti-HY5 western blot from wild-type and

hy5-1 seedlings grown for 7 d in continuous white light GL (100 mmol

quanta m22 s21) and wild type exposed to HL for 3 h.

Supplemental Table S1. Genes where expression changed at least 2-fold

following a shift from growth light (100 mmol photons m22 s21) to HL

in wild type.

Supplemental Table S2. Genes where expression changed at least 2-fold

following a shift from GL to BL in wild type.

Supplemental Table S3. Genes expressed at least 2-fold differently in the

cry1 mutant compared to wild type in GL conditions.


hy5 mutant compared to wild type in GL conditions.


cry1 mutant compared to wild type in HL conditions and not differ-

ently expressed in GL conditions.


hy5 mutant compared to wild type in HL conditions and not differently

expressed in GL conditions.

ACKNOWLEDGMENTS

We thank Dr. Markus Schmid for help with the microarrays. Dr. Vaughan

Hurry is acknowledged for critically reading the manuscript.

Received February 21, 2007; accepted May 1, 2007; published May 3, 2007.

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