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First published online May 3, 2007; 10.1104/pp.107.098293

Plant Physiology 144:1391-1406 (2007)
© 2007 American Society of Plant Biologists

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ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

Genome-Wide Gene Expression Analysis Reveals a Critical Role for CRYPTOCHROME1 in the Response of 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


   ABSTRACT
TOP
 ABSTRACT
RESULTS
 DISCUSSION
 CONCLUSION
 MATERIALS AND METHODS
 LITERATURE CITED
 
Exposure to high irradiance results in dramatic changes in nuclear gene expression in plants. However, little is known about the mechanisms by which changes in irradiance are sensed and how the information is transduced to the nucleus to initiate the genetic response. To investigate whether the photoreceptors are involved in the response to high irradiance, we analyzed expression 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. Following exposure to high intensity white light for 3 h (1,000 µmol quanta m–2 s–1) expression of ELIP1/2 and APX2 was strongly induced 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 the misregulation of these genes, the cry1 mutant displayed a high irradiance-sensitive phenotype with significant photoinactivation of photosystem II, indicated by reduced maximal fluorescence ratio. Thus, we describe a novel function of CRY1 in mediating plant responses to high irradiances that is essential to the induction of photoprotective mechanisms. This indicates that high irradiance can be sensed in a chloroplast-independent manner by a cytosolic/nucleic component.

Light is not only the primary energy source for plants but it also provides them with information to modulate developmental processes such as seed germination, seedling establishment, phototropism, chloroplast movement, shade avoidance, circadian rhythms, and flowering time (Fankhauser and Staiger, 2002Go; Chen et al., 2004Go). Plants can detect almost all facets of light, including direction, duration, and wavelength using three major classes of photoreceptors: the red/far-red light-absorbing phytochromes, the blue/UV-A light-absorbing cryptochromes and phototropins, and the UV-B-sensing UV-B receptors (Chen et al., 2004Go). These photoreceptors perceive light signals and initiate intracellular signaling pathways involving proteolytic degradation of signaling components and large reorganization of the transcriptional program to modulate plant growth and development (Chen et al., 2004Go).

In photosynthesis, light energy is absorbed by the light-harvesting antennae and converted into chemical energy by the reaction centers. However, when photon fluence exceeds the photon utilization capacity of the chloroplast, photosynthesis becomes photoinhibited and the reaction centers, particularly PSII, become irreversibly damaged and require repair (Aro et al., 1993Goa, 1993Gob). Furthermore, elevated excitation pressure has been demonstrated to increase the production of reactive oxygen species (ROS; Karpinski et al., 1997Go; Huner et al., 1998Go; Foyer and Allen, 2003Go), and the damaging effects of ROS include oxidation of lipids, proteins, and enzymes necessary for proper function of the chloroplast and the cell as a whole (Foyer and Allen, 2003Go). To protect themselves against extensive damage, plants have the ability to sense when photon fluence exceeds the photon utilization capacity of the chloroplast and communicate this information to stimulate changes in nuclear and chloroplast gene expression. Recent microarray experiments have revealed that expression of a large number of nuclear-encoded genes is affected by exposure to high irradiance (Rossel et al., 2002Go; Kimura et al., 2003Go; Richly et al., 2003Go; Vanderauwera et al., 2005Go). The mechanisms by which excess irradiance is sensed and how the information is transduced to the nucleus to initiate a genetic response are unknown, but it is well established that the redox state of the plastoquinone electron carrier pool is correlated with the expression of photosynthetic genes encoded in both the chloroplast and the nucleus (Escoubas et al., 1995Go; Huner et al., 1998Go; Karpinski et al., 1999Go; Pfannschmidt et al., 1999Go, 2001Go; Pfannschmidt, 2003Go). Furthermore, a unique light- and redox-controlled protein phosphorylation system has evolved in plant thylakoid membranes where intrinsic protein kinases are activated by light or reducing conditions and subsequently phosphorylate the membrane proteins of PSII and its light-harvesting antenna, light-harvesting complex II (LHCII; Vener et al., 1998Go). The phosphorylation state of these proteins has been suggested to be involved in the regulation of LHC expression in the nucleus (Rintamaki et al., 1997Go). In addition, under high irradiance conditions where the equilibrium between different ROS (e.g. hydrogen peroxide and OH·) production and scavenging is perturbed, subsequent changes in concentrations or rates of ROS production could also be initiators of signaling pathways originating in the chloroplast (Karpinski et al., 2003Go; Apel and Hirt, 2004Go).

Despite extensive work, the mechanisms by which excess irradiance is sensed and how the information is transduced to the nucleus to initiate a genetic response have remained elusive. Are plastid signals alone responsible for the regulation of nuclear gene expression in response to excess irradiance? The dramatic impact on nuclear gene expression by exposure to high intensity white light suggests that several signaling pathways are involved. Hence, we wanted to test whether components known to control photomorphogenesis such as cryptochromes (CRY1 and 2), phytochromes (PHYA and B), and two of their downstream transcription factors (LONG HYPOCOTYL5 [HY5] and HY5 HOMOLOG [HYH]) also are involved in the response to excess irradiance. We tested the expression of genes encoding EARLY LIGHT-INDUCIBLE PROTEIN1 and 2 (ELIP1 and ELIP2), ASCORBATE PEROXIDASE2 (APX2), and LIGHT-HARVESTING CHLOROPHYLL A/B-BINDING PROTEIN2.4 (LHCB2.4) after high intensity white light treatment in the cry1, cry2, phyA, phyB, phyAphyB, hy5, and hyh mutants. The cry1 and hy5 mutants showed misregulation of ELIP1/2 in response to high intensity white light, and by using the Affymetrix Arabidopsis (Arabidopsis thaliana) 24 K Gene-Chip representing 24,000 genes, we could demonstrate that a large group of the HL-responsive genes were regulated via a CRY1-mediated response and that 26 of those genes were also HY5 dependent. Our study demonstrates a novel function of CRY1 as a mediator of plant response to changes in irradiance and provides new insight into the high light stress-responsive transcriptome.


   RESULTS
TOP
 ABSTRACT
 RESULTS
DISCUSSION
 CONCLUSION
 MATERIALS AND METHODS
 LITERATURE CITED
 

Exposure to High Light Results in Adaptive Changes of the Transcriptome

Arabidopsis seedlings were grown for 7 d at 100 µmol quanta m–2 s–1 continuous white light (growth light [GL]) and exposed for 3 h to a high intensity white light treatment of 1,000 µmol quanta m–2 s–1 (HL). Exposure to HL results in significant light stress, as shown by the gradual drop in the variable to maximal fluorescence ratio (Fv/Fm) following exposure (Table I ). Long-term exposure (12 h) to HL results in a drop in Fv/Fm from 0.83 to 0.59 in wild type, indicative of PSII photoinactivation (Table I). To get a robust gene set responding to these experimental conditions, wild-type samples from three independent biological experiments were hybridized to ATH1 Genome Arrays (Affymetrix). Differentially expressed genes were identified with a combination of logit-t (Lemon et al., 2003Go) and the Filter on Fold Change tool in GeneSpring 7.3 (Agilent Technologies; Schmid et al., 2003Go). A total of 992 genes showed more than 2-fold change in expression in response to 3-h HL treatment. Thus, approximately 4% of all genes represented on the chip demonstrated changes in expression in response to HL, 660 genes were 2-fold up-regulated, and 332 were 2-fold down-regulated (Supplemental Table S1).


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  		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 of white light at 1,000 µmol of photons m–2 s–1. Measurements were made in humidified air. Each point is the mean ± SE of four to six leaves from four to six individual plants after 60-min dark acclimation.

 
Under our experimental conditions, ELIP1 and ELIP2 showed a very strong induction of expression, 100-fold and 88-fold, respectively, in response to 3-h HL treatment. As described previously, ELIP1 was more strongly induced by HL compared to ELIP2 (Heddad et al., 2006Go). APX2 was induced 6-fold and LHCB2.4 was reduced to less than 25% of the control. The ELIP1, ELIP2, APX2, and LHCB2.4 genes all demonstrated a robust response to our HL exposure and they all encode key components of photosynthetic light stress response. In addition, the observed response to HL of these genes is well documented in plants exposed to somewhat different experimental, high irradiance conditions (Adamska et al., 1992Go; Karpinski et al., 1999Go; Kimura et al., 2001Go; Rossel et al., 2002Go; Vanderauwera et al., 2005Go; Heddad et al., 2006Go). Thus, these four genes were selected as marker genes for the HL response in our further analysis using the photoreceptor mutants cry1, cry2, phyA, phyB, and phyAphyB and the transcription 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 irradiance, we analyzed ELIP1/2, APX2, and LHCB2.4 expression in the cryptochrome mutants cry1 and cry2 and the phytochrome mutants phyA, phyB, and phyAphyB. Seven-day-old wild-type and mutant seedlings grown in continuous white light (100 µmol quanta m–2 s–1) were exposed to high irradiance conditions identical to the conditions used for the microarray experiment (HL). In wild type, HL exposure resulted in a strong induction of ELIP1 (55-fold) and ELIP2 (50-fold), respectively (Fig. 1, A and B ). Thus, the strong induction observed in the microarray experiment was confirmed by real-time PCR. In the cry2 and phyA mutants, the induction of ELIP1 and ELIP2 expression was similar to that in wild type, whereas in the phyB and phyAphyB mutants, the induction of ELIP1 expression was somewhat lower than that in wild type (Fig. 1, A and B). In contrast, the cry1 mutant showed a strongly reduced induction of both ELIP1 (11-fold) and ELIP2 (8-fold) relative to wild type (55- and 50-fold, respectively; Fig. 1, A and B).


Figure 1
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  		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 of ELIP1 (A; At3g22840), ELIP2 (B; At4g14690), APX2 (C; APX1b, At3g09640), and LHCB2.4 (D; At3g27690) was analyzed using real-time PCR. The fold induction after HL exposure related to the control of each genotype is presented. The results were normalized to the expression level of At4g36800 encoding a ubiquitin-protein ligase-like protein. The mean ± SE of at least four biological replicates is shown.

 
The basic Leu zipper transcription factor HY5 is involved in the promotion of light-induced gene expression (Ang et al., 1998Go; Chattopadhyay et al., 1998Go). ELIP1 and ELIP2 expression levels were investigated in the hy5 mutant and in a T-DNA knockout line of the HY5 homolog HYH, hyh. In the hy5 mutant, both ELIP1 and ELIP2 induction was suppressed to levels similar to those observed in the cry1 mutant, whereas the hyh mutant demonstrated wild-type induction (Fig. 1, A and B). APX2 was induced in all investigated mutants after HL treatment (Fig. 1C). LHCB2.4 expression in the wild type was reduced to 25% of the control level after HL treatment (Fig. 1D) and similarly reduced in all investigated mutants. Thus, our results demonstrate that a significant part of the induction of ELIP1 and ELIP2 in response to high irradiance is mediated via CRY1 and HY5. The misregulation of ELIP1 and ELIP2 in response to HL exposure observed in the cry1 and hy5 seedlings was also observed in 5-week-old plants (data not shown).


ELIP1/2 Expression Is Strongly Induced by High Intensity Blue Light

The mutant analysis demonstrated that the blue light receptor CRY1 is involved in the HL-induced expression of ELIP1 and ELIP2. Consequently, we analyzed the response of ELIP1/2 in different intensities of blue light (400–540 nm). Wild-type seedlings were grown for 7 d in continuous white light of 100 µmol quanta m–2 s–1 (10 µmol quanta m–2 s–1 blue light) and were subjected to 3 h of 25, 50, 100, and 200 µmol quanta m–2 s–1 blue light. ELIP1 and ELIP2 induction increased gradually with increasing intensities of blue light (Fig. 2 ). Following an increase in irradiance from 10 to 25 µmol quanta m–2 s–1, ELIP1 and ELIP2 were induced 3- and 3.5-fold, respectively, and when transferred from 10 to 200 µmol quanta m–2 s–1 blue light (BL), ELIP1 and ELIP2 induction increased to 27- and 37-fold, respectively. Our results support published data using etiolated seedlings demonstrating a blue light intensity-dependent accumulation of ELIP transcript (Adamska, 1995Go). However, after transfer of the cry1 mutant from 10 to 200 µmol quanta m–2 s–1 BL, ELIP1 was only 3-fold induced, and the ELIP2 induction was abolished. ELIP1 and ELIP2 expression was not induced by high intensity red light in wild type (300 µmol quanta m–2 s–1; data not shown). Taken together, these data indicate that the HL-induced expression of ELIP1/2 is predominantly mediated via CRY1 in a blue light intensity-dependent manner.


Figure 2
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  		Figure 2. Real-time analysis of ELIP1/2 expression at different blue light intensities. Wild-type and cry1 seedlings were grown for 7 d in continuous 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 after BL exposure related to the control of each genotype is presented. The results were normalized to the expression level of At4g36800 encoding a ubiquitin-protein ligase-like protein. The mean ± SE of at least four biological replicates is shown.

 

CRY1 and HY5 Regulate a Large Number of Genes in Response to High Light

To determine the identities of the CRY1 and HY5 regulons in response to high irradiance, we performed expression profile analysis of cry1 and hy5 mutants in response to HL to be compared with the expression profile of wild type exposed to HL. In addition, we analyzed the expression profile analysis of wild type in response to BL. Seedlings were grown at 100 µmol quanta m–2 s–1 continuous white light and then subjected to BL (wild type) or to HL for 3 h (wild type, cry1, and hy5). Samples from three independent biological experiments were hybridized to ATH1 Genome Arrays (Affymetrix). Wild-type, cry1, and hy5 seedlings grown and kept at 100 µmol quanta m–2 s–1 continuous white light (GL) were used as controls.

Analysis of the control, GL-grown seedlings demonstrated that 48 genes were differentially expressed in the CRY1-deficient seedlings (Supplemental Table S3), and 290 genes were differently expressed in the HY5-deficient seedlings (Supplemental Table S4) compared to wild type. Expression analysis of the cry1 and hy5 mutants during early light development or following a shift from darkness to light has been performed previously (Ma et al., 2001Go; Holm et al., 2002Go; Folta et al., 2003Go; Jiao et al., 2003Go; Ohgishi et al., 2004Go; Ulm et al., 2004Go). However, a direct comparison between our experiment and published results is difficult due to the usage of very different plant material. The 48 genes differentially expressed in the cry1 mutant compared to wild type under our growth conditions were classified into different categories according to The Arabidopsis Information Resource (TAIR) gene ontology (http://www.arabidopsis.org/tools/bulk/go/index.jsp). A large proportion of those genes encoded proteins with unknown function. Three genes encoding redox-related proteins, peroxidase (At2g41480), an electron carrier (At5g44440), and glutathione dehydrogenase (At1g75270), were identified as genes misregulated in the cry1 mutant. Only three of the 48 genes differently expressed in the cry1 mutant encode transcription factors (At1g75240, At2g33860, and At5g60450; Supplemental Table S3).

In the hy5 mutant, as many as 290 genes (Supplemental Table S4) were differentially expressed compared to wild type. Among these 290 genes were 14 transcription factors (At5g25190, At5g39860, At1g52830, At5g53980, At1g21910, At2g14210, At3g15540, At3g58120, At1g35560, AT5g25810, At2g47460, At2g17040, and At5g07690) and HY5 (At5g11260) was one of them (Supplemental Table S4). Several genes encoding enzymes in the phenylpropanoid pathway, CHALCONE SYNTHASE (CHS), FLAVANONE 3-HYDROXYLASE (F3H), and FLAVONOL SYNTHASE1 (FLS1), were differently expressed in hy5 compared to wild type (Supplemental Table S4). ELIP1 was more than 2-fold differentially expressed in hy5 compared to wild type under the control conditions (Supplemental Table S4), and HY5 has previously been shown to be involved in the induction of ELIP1 and establishment of PSII activity during a dark-to-light transition (Harari-Steinberg et al., 2001Go).

The genes that showed a change in expression in the hy5 and cry1 mutants under control conditions were excluded from our further analysis, and we focused only on the genes that changed in wild type after HL treatment (992 genes) and that were at least 2-fold differentially expressed after HL treatment in the cry1 or hy5 mutants compared to wild type. Only four genes were excluded from the HL list due to misregulation 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 were misregulated in the cry1 and hy5 in response to HL, respectively (Fig. 3A; Supplemental Tables S5 and S6). The overlap between genes misregulated in response to HL in both cry1 and hy5 was 26 genes (Fig. 3A).


Figure 3
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  		Figure 3. Flow chart demonstrating the analysis used to filter genes that were misregulated in cry1 and hy5 compared to wild type after HL (A) and BL (B) exposure.

 
To understand the CRY1 regulon, we analyzed the expression profile of wild-type seedlings exposed to BL. Using the same cutoffs as for the HL microarrays (Supplemental Table S1), 858 genes changed 2-fold or more after exposure to BL in wild type (Fig. 3B). When we compared the expression profiles of wild-type plants exposed to HL and BL, the overlap was 369 genes; 225 genes 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 to HL and BL in wild type (369 genes) and the misregulated genes in cry1 or hy5 was 49 and 38 genes, respectively (Table II ; Fig. 3B). Expression of 23 of the 26 cry1 and hy5 HL-misregulated genes also changed in response to high intensity BL in wild type (Table II; Fig. 3B). Thus, it is clear that CRY1 and HY5 are necessary for the regulation of a significant group of genes responding to high irradiance (Table II; Fig. 3). The 49 and 38 genes misregulated in cry1 and hy5, respectively, were classified into different categories according to the TAIR gene ontology (http://www.arabidopsis.org/tools/bulk/go/index.jsp; Fig. 3B; Table II). The dominant groups of genes that were misregulated in the mutants encode components involved in transcriptional regulation, enzymes involved in the phenylpropanoid pathway, proteins associated with stress responses, and proteins with unknown function (Table II).


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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.

 
Expression of HY5 is induced by HL exposure and the response is dependent on CRY1. The genes encoding the transcription factors PAP1 (PRODUCTION OF AN-THOCYANIN PIGMENT1; MYB75 and At1g56650) and PAP2 (MYB90 and At1g66390; Borevitz et al., 2000Go) involved in the regulation of components in the flavonoid biosynthesis were misregulated in cry1 and in cry1 and hy5, respectively. The genes encoding components catalyzing the entry of metabolites into the phenylpropanoid pathway, such as CHORISMATE MUTASE1 (CM1; At3g29200) and PHE AMMONIA-LYASE1 (PAL1; At2g37040) and PAL2 (At3g53260), were found to be strongly induced by BL in wild type and misregulated in the cry1 and hy5 mutants (Table II; Supplemental Fig. S1). In the middle section of the pathway, expression of genes encoding 4-COUMARATE:CoA LIGASE3 (4CL3; At1g65060) and CHALCONE-FLAVANONE ISOMERASE (CHI; At3g55120) was inhibited in both cry1 and hy5 mutants (Table II; Supplemental Fig. S1). In addition, genes encoding the components 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 GLUTATHIONE PEROXIDASE7 (GPX7) gene (At4g31870), were strongly up-regulated in wild type in response to HL and BL, and the induction of GPX7 was suppressed in cry1 and hy5. Furthermore, the expression of At1g10370 and At1g02940, both encoding glutathione S-transferases (GSTs), was misregulated in cry1 and hy5, respectively (Table II). Genes encoding further potential components in stress response such as ubiquinone methyltransferase (At2g41040) and PYRIDOXIN BIOSYNTHESIS2 (PDX2; At5g60540) were also misregulated in response to HL in the cry1 mutant (Table II). The microarray analysis also confirmed the suppressed induction of ELIP1/2 in the cry1 and hy5 mutants shown with real-time PCR. However, ELIP1 is not grouped as misregulated in hy5 (Table II) because it was already more than 2-fold differentially expressed in hy5 under the control growth conditions.


CRY1 and HY5 Regulate HL-Responsive Genes via G-Box Promoter Elements

HY5 has previously been demonstrated to bind a G-box motif (Gao et al., 2004Go). We examined the 500-bp promoter sequences of all cry1 and/or hy5 HL-misregulated genes (116 in total [77 + 65]) to determine the frequency of occurrence of the G-box consensus (Fig. 3A). Within the promoters of the 26 genes misregulated in both cry1 HL and hy5 HL (Fig. 3A), a classic G box (CACGTG) was found in five promoters (19%; Table III ). The G-box frequency in the hy5-specific HL regulon was 23% (9/39 genes; Table III). These numbers represented a significant enrichment versus the genomic average for classic G-box elements within Arabidopsis 500-bp promoter spaces (10.3% in the TAIR annotated Arabidopsis genome, v.6). Inclusion of G-box sequence variants increased these frequencies to 35% (14/26 cry1 + hy5 HL overlap) and 56% (22/39 hy5-specific HL genes), respectively (Table III). Furthermore, in the 51 genes specific to the cry1 HL regulon (Fig. 3C), 16 (31%) promoters contained a classic G box (CACGTG), and addition of the G-box variants (CACGTH) collectively accounted for another 24% (12/51) of the cry1-specific HL gene set. Thus, the CACGTH frequency was 55% in cry1-specific HL gene regulon (Table III).


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  		Table III. Known and novel cis-regulons demonstrating significant ({chi}2, P < 0.05) transcriptional responses during exposure HL and BL in wild-type, cry1, and hy5 Arabidopsis backgrounds

{chi}2 expected values were calculated based on the whole-array response frequencies. ind, Induced genes; rep, repressed genes.

 
The CRY1 and HY5 regulons identified in our array experiments using stringent statistical selection criteria (logit-t P < 0.025 and uniform 2-fold change in expression) were small and therefore prohibitive for further bioinformatic analyses such as in silico mutagenesis and novel cis-element detection. Subsequent cis-regulon analyses of our microarray data therefore used sliding-scale fold-change standards in combination with a novel motif bioactivity-testing protocol (Benedict et al., 2006Go; Geisler et al., 2006Go). The algorithm used reduces false positives by correlating the naturally occurring distribution of cis-elements in the Arabidopsis genome with the specific HL or cry1- and hy5-responsive genes (Geisler et al., 2006Go). Using the new fold-change criteria, the total size of the wild-type HL differentially expressed gene regulon increased to 1,577 genes (820 induced and 757 repressed), the wild-type BL regulon increased to 1,516 genes (634 induced and 882 repressed), the cry1 HL-misregulated gene regulon increased to 371 genes (217 induced and 154 repressed compared to wild type), and the hy5 HL-misregulated gene regulon increased to include 707 genes (348 induced and 359 repressed compared to wild type). Genes containing the classic G box within their 500-bp promoter demonstrated a strong overrepresentation in the wild-type HL- and BL-induced regulons (Table III). The G box was also overrepresented in the regulons of genes repressed in cry1 and hy5 HL-treated seedlings compared to wild type. By performing in silico mutagenesis (Benedict et al., 2006Go; Geisler et al., 2006Go) of the classic G-box sequence in wild-type HL, hy5 HL, and cry1 HL backgrounds, we were able to establish that the palindromic G-box consensus could vary at position 1 (or inversely, position 6) without loss of bioactivity (Supplemental Fig. S3).

A more general survey of cis-elements previously reported to contribute to light-, phytochrome-, cryptochrome-, and HY5-regulated transcription (PLACE database; http://www.dna.affrc.go.jp/PLACE; Gao et al., 2004Go) showed that the promoters of genes differentially regulated during BL and HL treatment of wild type, and HL treatment of cry1 and hy5 seedlings, were also enriched for the I-box and myelocytomatosis oncogene (MYC) cis-elements. The respective cis-regulons were significantly more likely to be induced or repressed in response to HL than the general population of genes on the microarray (Table III). In an attempt to identify novel cis-elements, we used the Gibbs sampling-based Inclusive Motif Sampler program (Thijs et al., 2002Go) to identify 8- to 10-bp consensus sequences overrepresented in the 500-bp promoters of the genes associated with the CRY1- and HY5-mediated transcriptional responses to HL (Supplemental Tables S5 and S6). Excluding hits resembling the G box, the most significant cry1 HL-repressed, regulon-enriched element was GnTCKAG (CryR1; Table III). Bioactivity of CryR1 was indicated by the significant overrepresentation in the promoters of genes induced by HL in wild type. Another element identified in the list of cry1 HL-induced genes was ACATAwCT (CryR2; Table III). Bioactivity of the CryR2 element was also indicated by a significant repression at frequencies greater than that predicted by random chance of the genes where the promoter contains the element following HL and BL treatments in wild type. In addition, in silico mutagenesis of the CryR1 and CryR2 elements demonstrated that the CryR1 element could not vary without losing biological activity, whereas the CryR2 element could vary only at position 2 without loss of bioactivity (Supplemental Fig. S4). Thus, genes containing the CryR1 (GnTCKAG) and the two CryR2 variants (ACATAwCT and ADATAwCT) were all significantly induced and repressed, respectively, in response to HL in wild type (Supplemental Fig. S4). Furthermore, genes containing these elements were repressed or induced in the cry1 mutant compared to wild type (Supplemental Fig. S4). Another element, HycR1 (ACmyACAy), was identified from the cry1 HL-induced gene regulon, and bioactivity was confirmed in cry1 and hy5 HL-treated plants (with significant cis-regulon enrichment in the induced gene groups for these mutants; Table III).


The cry1 and hy5 Mutants Demonstrate a Defective Stress Response

In wild type, anthocyanin accumulation increased with prolonged exposure to high irradiance, and after 24-h exposure, more than a 3-fold increase was observed (Supplemental Fig. S2). In contrast, anthocyanin contents were unchanged in the cry1 and hy5 mutants after 9-h exposure to HL (Supplemental Fig. S2), supporting the misregulation of genes encoding components of the phenylpropanoid pathway observed in cry1 and hy5 (Supplemental Fig. S1). Reduced anthocyanin levels in CRY1-deficient Arabidopsis seedlings have been shown in continuous blue light (Ahmad et al., 1995Go; Lin et al., 1996Go) and in continuous white light (Neff and Chory, 1998Go). However, our results demonstrate that CRY1 and HY5 also play a key role in the high irradiance-induced accumulation of anthocyanin. HY5 is subject to regulation via COP9 signalosome-mediated degradation in the dark (Hardtke et al., 2000Go; Osterlund et al., 2000Go). To verify that HY5 protein itself was stable under HL conditions, western blots were performed, and no reduction in HY5 protein could be detected in HL samples compared to LL samples (Supplemental Fig. S5).

Exposure to HL resulted in a gradual photoinactivation of PSII as demonstrated by a drop in Fv/Fm from 0.83 to 0.73 after only 3 h exposure and further down to 0.59 after 12 h in wild type (Table I). Despite the reduced accumulation of anthocyanin in the hy5 mutant, the sensitivity to HL exposure was similar to wild type in the mutant (Table I). In contrast, the cry1 mutant was twice as sensitive to the HL exposure as wild type, shown by a drop in Fv/Fm from 0.83 to 0.32 (Table I). Furthermore, photo bleaching after prolonged (24 h) exposure to HL was clear in the cry1 mutant compared to wild type and the hy5 mutant, both in seedlings and in 5-week-old plants (Fig. 4 ). The HL-sensitive phenotype of the cry1 mutant supports the conclusion from the CRY1 regulon that a significant component of the HL photoprotective response is mediated by CRY1.


Figure 4
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  		Figure 4. Representative photographs of 5-week-old plants of the cry1 mutant (A) and wild type (B) following 24-h exposure to HL. Photographs of 7-d-old seedlings of the cry1 mutant (C) and wild type (D and E) following 24-h exposure to HL. F, Chlorophyll content in control-grown and 24-h HL-exposed seedlings of the cry1 mutant and wild type. The mean ± SD of four biological replicates is shown. One-way ANOVA followed by Bonferroni's multiple comparison test to compare column pairs (wild-type control versus wild-type HL and cry1 control versus cry1 HL) was performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, www.graphpad.com). ***, P < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
CONCLUSION
 MATERIALS AND METHODS
 LITERATURE CITED
 
The role of cryptochromes during early light response (hours of light exposure) has been well established, and expression profiles of the Arabidopsis cry1 mutants revealed that a large number of genes are regulated via cryptochromes (Ma et al., 2001Go; Folta et al., 2003Go; Jiao et al., 2003Go; Ohgishi et al., 2004Go). However, in 7-d-old seedlings grown at 100 µmol quanta m–2 s–1, only 48 genes were differentially regulated in the cry1 mutant compared to wild type (Supplemental Table S3). Exposure of the cry1 mutant to HL revealed that 77 HL-responsive genes were differentially expressed in the mutant compared to wild type exposed to HL (Fig. 3A). Furthermore, 49 of the 77 HL genes misregulated in the cry1 mutant were also induced in wild type by high intensity BL (Fig. 3B). The remaining 28 CRY1-dependent genes require light qualities outside the narrow waveband used in these BL experiments, possibly through functional interaction between cryptochrome and phytochromes (Ahmad et al., 1998bGo; Mas et al., 2000Go). Thus, a large number of genes specific to the high light response were discovered to be mediated via the photoreceptor CRY1, and we have described a novel function of CRY1 as a mediator of plant responses to high irradiance controlling the expression of a large number of HL-responsive genes.

During exposure to high irradiance, CRY1 promotes the expression of genes associated with stress protection mechanisms such as GPX7, encoding a putative glutathione peroxidase (At4g31870) and the GST ERD9 (At1g10370; Table II). The GST proteins have been shown to respond to various stresses such as HL, cold, and drought (Wagner et al., 2002Go; Seki et al., 2003Go; Goulas et al., 2006Go). After exposure to HL, six different GST genes were induced, and four of them were induced in both HL and high intensity BL (Supplemental Tables S1 and S2). Thus, our results suggest that GSTs play an important role in the response to high light stress. In addition, genes encoding a potential ubiquinone methyltransferase (At2g41040) and PDX2 (At5g60540) are misregulated in response to HL in the cry1 mutant (Table II). The gene At2g41040 has a predicted ubiquinone methyltransferase domain and the encoded protein was recently reported to be found in plastoglobuli (Vidi et al., 2006Go; Ytterberg et al., 2006Go) and could potentially be involved in phylloquinone (vitamin K1) biosynthesis. Phylloquinones serve as electron acceptors in the PSI reaction center and are critical for photosynthetic function. PDX2 together with PDX1 form a Gln amidotransferase complex involved in vitamin B6 biosynthesis (Tambasco-Studart et al., 2005Go). Vitamin B6 has been shown to be a potent antioxidant with the ability to quench ROS such as singlet oxygen and superoxide in human erythrocytes (Jain and Lim, 2001Go) and fungus (Ehrenshaft et al., 1999Go) and prevents lipid peroxidation in human erythrocytes (Jain and Lim, 2001Go). Presumably, vitamin B6 could also protect photosynthetic membranes against lipid peroxidation caused by ROS production during high light stress. Taken together, these data indicate an important role of CRY1 in modulating the response of plants to changes in irradiance leading to oxidative damage.

Blue light receptors have been described to regulate a range of different plant responses, including deetiolation, photo entrainment of the circadian clock, phototropic curvature, and chloroplast relocation. In addition to CRY1, three different blue light receptors with known functions have been described in Arabidopsis, CRY2, and the phototropins PHOT1 and PHOT2. The cry2 mutant did not show impaired expression of our HL marker genes after exposure to high irradiance (Fig. 1). The large number of genes that were misregulated in response to HL in the cry1 mutant excludes redundant roles of CRY1 and CRY2 under the HL conditions. The underlying reason for this may be found in the different behavior of the two proteins; the CRY2 protein is rapidly degraded by UV-A, blue, and green light, whereas the CRY1 protein is stable (Ahmad et al., 1998aGo; Lin et al., 1998Go). CRY2 is particularly important in retarding hypocotyl growth in response to low intensity blue light, whereas CRY1 has a prevalent role in response to higher blue light intensity (Lin et al., 1998Go). Furthermore, CRY1 phosphorylation kinetics and activation exhibits a positive correlation with blue light intensity in direct contrast to CRY2, which exhibits a negative correlation with blue light intensity (Shalitin et al., 2002Go, 2003Go). Thus, although CRY1, but not CRY2, has been shown to mediate a response to higher irradiances, a role for CRY1 during changes in irradiance and the high light response has not been previously described. Related to this proposed role of CRY1 in mediating light stress response, the phototropin PHOT2 was shown to regulate the avoidance movement of chloroplasts in response to high intensity blue light (Jarillo et al., 2001Go; Kagawa et al., 2001Go; Sakai et al., 2001Go). In low light, chloroplasts are distributed along the periclinal walls in a manner that presumably maximizes light capture for photosynthesis, whereas under high fluence rates, chloroplast damage is minimized by movement of the chloroplasts toward the anticlinal walls (Jarillo et al., 2001Go). This raises the possibility that the CRY1 and PHOT2 responses share similar mechanisms. However, earlier studies have shown that the cry1cry2 double mutant shows normal blue light-induced chloroplast relocation (Kagawa and Wada, 2000Go), indicating that these two classes of photoreceptors, CRY1 and PHOT2, act through separate pathways to provide protection against excess light.

Twenty-six genes of the 77 CRY1-dependent genes were also misregulated in response to HL in the hy5 mutant, and 23 of those 26 genes are also regulated by BL in wild type (Fig. 3, A and B). HY5 is transcriptionally activated in a phytochrome-dependent manner in etiolated seedlings exposed to light (Tepperman et al., 2001Go). Interestingly, expression of HY5 and the amount of HY5 protein was induced by HL exposure (Table II; Supplemental Fig. S5), and the response to HL was dependent on CRY1 activity (Table II). The expression profiles of cry1 and hy5 demonstrated that CRY1 and HY5 are connected in the response to HL but that they also play distinct roles (Fig. 5 ). Genes encoding enzymes associated with the phenylpropanoid pathway are represented in the group of genes misregulated in both cry1 and hy5 mutants (Table II). Expression of genes encoding the CHS has previously been demonstrated to be regulated by blue light in a CRY-dependent manner (Fuglevand et al., 1996Go). However, we demonstrate here that genes encoding components at every level of the phenylpropanoid pathway, from transcriptional control to the final enzymatic 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 accumulation could be detected in the hy5 seedlings (Supplemental Fig. S2). Furthermore, the induction of ELIP1/2 and UGT84A1 was impaired in the cry1 and hy5 mutants following exposure to HL. It has previously been demonstrated that the loss of HY5 impairs the light-induced expression of ELIP1 (Harari-Steinberg et al., 2001Go) and the UV-B-responsive expression of several genes such as ELIP1/2 and UGT84A1 (Ulm et al., 2004Go; Brown et al., 2005Go). In addition to the effect on gene expression following exposure to UV-B, hy5 demonstrated a UV-B sensitive phenotype, and HY5 was found to be a key component of the UVR8 pathway required for survival under UV-B radiation (Brown et al., 2005Go; Oravecz et al., 2006Go). The overlap between the genes misregulated in the hy5 mutant compared to wild type, in response to UV-B, and to high intensity blue light was small (only five genes), indicating that the UV-B and HL responses are triggered by two separate mechanisms. Thus, our results demonstrate that in addition to a role in photomorphogenesis and in the UV-B response, HY5 is a key component of the CRY1-mediated pathway in response to HL. In addition, 39 genes were misregulated in the hy5 mutant in response to HL in a CRY1-independent manner (Fig. 5). Two genes encoding components related to pathogen defense signaling ELI3-2 (At4g37990) and a WRKY family transcription factor, WRKY70 (At3g56400; Kiedrowski et al., 1992Go; Li et al., 2004Go), are misregulated in the hy5 mutant (Supplemental Table S6), suggesting that HY5 may be a key component of the cross talk between light and pathogen defense signaling. HY5 has been demonstrated to bind a G-box motif (Gao et al., 2004Go), and, consistent with this, bioinformatic analysis of our array data revealed that a classic G-box (CACGTG) or a G-box variant (CACGTH) were significantly enriched in the HY5 regulon versus the genomic average for these elements within Arabidopsis 500-bp promoter spaces (Table III).


Figure 5
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  		Figure 5. Working model for the CRY1-mediated HL response.

 
A large number of genes encoding components necessary for the photoprotective response in plants are misregulated in the cry1 mutant. The effect of this limited response was shown in the cry1 mutant with the 60% loss of Fv/Fm after a 12-h high light exposure compared to around 30% loss in hy5 and wild type (Table I). Furthermore, photo bleaching after prolonged (24 h) exposure to high light was exacerbated in the cry1 mutant compared to wild type both in seedlings and in 5-week-old plants (Fig. 4). Thus, the cry1 mutant has more difficulty recovering from high light exposure than wild type. However, the cry1 mutant was reported to be able to acclimate photosynthetic capacity to high light after long-term (weeks) exposure (400–600 µmol quanta m–2 s–1; Walters et al., 1999Go; Weston et al., 2000Go). However, Weston et al. (2000)Go demonstrated that a shift directly to 600 µmol quanta m–2 s–1 without a pretreatment with 400 µmol m–2 s–1 resulted in a severe light stress in the cry1 mutant compared to wild type. Even though a direct comparison with the results of Weston et al. (2000)Go cannot be made, our results confirm an essential role for CRY1 during high light stress response. Despite the large number of genes, including ELIP1/2 and components of the phenylpropanoid pathway found to be misregulated in both hy5 HL and cry1 HL, the tolerance to photoinhibition was not changed in hy5 (Table I). A double null ELIP Arabidopsis mutant did not demonstrate reduced tolerance to photoinhibition and photooxidative stress compared to wild type, indicating that the ELIP proteins do not have a photoprotective function (Rossini et al., 2006Go). The difference in light stress sensitivity between cry1 and hy5 mutants may be explained by the fact that CRY1 is the mediator of the high irradiance response and thereby controls a larger number of genes compared to the downstream component HY5 (Fig. 4). At this point, we can only speculate about which of the CRY1-specific genes encode key components of the protective mechanisms against light stress. However, it is worth noting that the MYB family transcription factor (At5g49330), At3g17610, encoding a protein with unknown function and the GSTs (ERD9) are all strongly induced in wild type by HL and BL and misregulated specifically in cry1.

Light-regulated protein degradation is central to cryptochrome signaling, and CRY1 was found to interact with the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1; Yang et al., 2001Go). COP1 is required for light-regulated degradation of several transcription factors involved in light-regulated transcription, including HY5. It was proposed that a light-driven conformational change of the cryptochromes induces a structural modification of COP1 that releases HY5 bound by COP1 in the dark (Cashmore, 2003Go). Furthermore, a crucial role for COP1 as a positive regulator of the UV-B response was recently demonstrated (Oravecz et al., 2006Go). It is possible that COP1 is involved in the CRY1-mediated high irradiance response we have reported here, and future work will demonstrate if the CRY1-COP1-HY5 signaling system that is used to regulate photomorphogenesis also plays a role in the response to high irradiances. Two novel cis-elements were enriched in the list of CRY1-dependent HL-responsive genes, CryR1 (GnTCKAG) and CryR2 (ACATAwCT; Table III). CryR1 was significantly enriched in the promoters of genes induced by HL in wild type, suggesting interaction with an activator of gene expression. In contrast, the CryR2 element was significantly enriched in the promoters of genes repressed by HL in wild type, suggesting interaction 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 elements are targets of the CRY1-mediated high irradiance transcriptional response.


   CONCLUSION
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 CONCLUSION
MATERIALS AND METHODS
 LITERATURE CITED
 
Analysis of the high irradiance response of the photoreceptor mutants phyA, phyB, cry1, and cry2 and the transcription factor mutants hy5 and hyh revealed a novel function of CRY1 in mediating plant responses to high irradiances. In addition to a role in photomorphogenesis, CRY1 is essential to the induction of photoprotective mechanisms against high light stress. Thus, we have demonstrated that high irradiance signals can be transduced in a chloroplast-independent manner by cytosolic/nucleic components.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 CONCLUSION
 MATERIALS AND METHODS
LITERATURE CITED
 

Plant Material and Growth Conditions

Seeds from Arabidopsis (Arabidopsis thaliana) Columbia-0 (Col-0) and phyA-211, phyB-9, cry1-304 (Ahmad and Cashmore, 1993Go), cry2-1 (Guo et al., 1998Go), and hy5 (Maxwell et al., 2003Go) 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 1x Murashige and Skoog basal salt mixture including vitamins (Duchefa) and 2% Suc. The plates were stratified 2 d in darkness at 4°C and then placed either 7 d into continuous white light (100 µmol quanta m–2 s–1, 23°C or were transferred 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 µmol quanta m–2 s–1 (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 directly frozen 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)Go. In brief, 50 to 70 mg seedlings were incubated overnight in 450 µL methanol acidified with 1% HCl. After the addition of 250 µL distilled water, anthocyanins were separated from chlorophylls with 625 µL chloroform. The anthocyanin content was determined by measuring A530 and A657 of the aqueous phase and subtracting 0.25 x 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)Go was used for the parameters of chlorophyll fluorescence. The maximal photochemical efficiency of PSII photochemistry in the dark-acclimated state was evaluated as Fv/Fm = (Fm – Fo)/Fm (van Kooten and Snel, 1990Go) after 1 h acclimation to darkness. In both the light- and dark-acclimated states, the minimal fluorescence intensity was measured by analytic modulated light, the maximal fluorescence intensity by saturating pulses (flash light intensity approximately 4,000 µmol photons m–2 s–1) 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 µg 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 µL of the diluted cDNA was used in a 20-µL iQ SYBR Green Supermix reaction (Bio-Rad). All reactions were performed in triplicate. The following primers were used: ELIP1 (At3g22840) forward primer, 5'-CGTTGCCGAAGTCACCAT-3', reverse primer, 5'-AATCCAACCATCGCTAAACG-3'; ELIP2 (At4g14690) forward primer, 5'-CACCACAAATGCCACAGTCT-3', reverse primer, 5'-TGCTAGTCTCCCGTTGATCC-3'; LHCB2.4 (At3g27690) forward primer, 5'-GCCATCCAACGATCTCCTC-3', reverse primer, 5'-TGGTCCGTACCAGATGCTC-3'; cytosolic APX2 (At3g09640) forward primer, 5'-CAAGGAGCTGTTCCCTATTCTG-3', reverse primer, 5'-GAGGTGGCTCAACTTTGTCC-3'; and ubiquitin-protein ligase-like protein (At4g36800) forward primer, 5'-CTGTTCACGGAACCCAATTC-3', reverse primer, 5'-GGAAAAAGGTCTGACCGACA-3'. The primers were designed to flank intron sites 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 s at 95°C, 30 s at 55°C, and 10 s at 72°C, after which a melting curve was performed. 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., 2003Go) and according to Pfaffl (2001)Go.


Microarray Analysis

cRNA Synthesis and Hybridization to Affymetrix GeneChips
RNA quality was assessed by agarose gel electrophoresis and spectrophotometry. 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 instructions (Affymetrix). In brief, 5 µg of total RNA was used in a reverse transcription 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 µg of fragmented, biotinylated cRNA was used for hybridization. Hybridization, washing, staining, and scanning procedures were performed as described in the Affymetrix technical manual. A Hybridization 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 and logit-t files were loaded into GeneSpring 7.3 (Agilent Technologies). Affymetrix 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 enrichment were performed as previously described (Benedict et al., 2006Go; Geisler et al., 2006Go). 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 (+1), or repressed (–1), using the sliding-scale fold-change standard described in Benedict et al. (2006)Go. 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, {chi}2 comparisons 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., 2002Go; http://homes.esat.kuleuven.be/~thijs/Work/MotifSampler.html) were also tested using the {chi}2 comparison of cis-regulon induction/repression frequency versus general array population induction/repression frequency.


Protein Extraction, Western Blot, and Chlorophyll Extraction

Proteins were extracted according to Hurry et al. (2000)Go and the SDS-PAGE according to Lundmark et al. (2006)Go. The HY5 antibody was provided by Santa Cruz Biotechnology. The chlorophyll was extracted and analyzed according to Porra et al. (1989)Go.


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 response 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 and shifted to HL for the time period indicated.
Supplemental Figure S3. In silico mutagenesis of the palindromic G-box binding consensus (wild type = 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 µmol quanta m–2 s–1) 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 µmol photons m–2 s–1) 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.
Supplemental Table S4. Genes expressed at least 2-fold differently in the hy5 mutant compared to wild type in GL conditions.
Supplemental Table S5. Genes expressed at least 2-fold differently in the cry1 mutant compared to wild type in HL conditions and not differently expressed in GL conditions.
Supplemental Table S6. Genes expressed at least 2-fold differently in the 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.


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

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

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Åsa Strand (asa.strand{at}plantphys.umu.se).

[W] The online version of this article contains Web-only data. Back

www.plantphysiol.org/cgi/doi/10.1104/pp.107.098293

* Corresponding author; e-mail asa.strand{at}plantphys.umu.se; fax 46–90–786–6676.


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 CONCLUSION
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