| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online May 3, 2007; 10.1104/pp.107.098293 Plant Physiology 144:1391-1406 (2007) © 2007 American Society of Plant Biologists
Genome-Wide Gene Expression Analysis Reveals a Critical Role for CRYPTOCHROME1 in the Response of Arabidopsis to High Irradiance1,[W]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 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, 2002  ; Chen et al., 2004). 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., 2004). 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., 2004).
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., 1993 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.
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., 2003
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., 2006 ). 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., 1992; Karpinski et al., 1999; Kimura et al., 2001; Rossel et al., 2002; Vanderauwera et al., 2005; Heddad et al., 2006). 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.
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).
The basic Leu zipper transcription factor HY5 is involved in the promotion of light-induced gene expression (Ang et al., 1998 ; Chattopadhyay et al., 1998). 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).
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, 1995
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., 2001
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., 2001 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).
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).
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., 2000 ) 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.
HY5 has previously been demonstrated to bind a G-box motif (Gao et al., 2004
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., 2006 ; Geisler et al., 2006). 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., 2006). 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., 2006; Geisler et al., 2006) 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., 2004
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., 1995 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.
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., 2001 ; Folta et al., 2003; Jiao et al., 2003; Ohgishi et al., 2004). 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., 1998b; Mas et al., 2000). 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., 2002
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., 1998a
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., 2001
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., 1999 ; Weston et al., 2000). However, Weston et al. (2000) 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) 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., 2006). 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., 2001
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.
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
Relative anthocyanin levels were determined according to Neff and Chory (1998)
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)
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 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., 2003
cRNA Synthesis and Hybridization to Affymetrix GeneChips
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.
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., 2006
Proteins were extracted according to Hurry et al. (2000)
The following materials are available in the online version of this article.
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.
1 This work was supported by the Swedish Research Foundation and Foundation for Strategic Research (INGVAR grant to Å.S.). 
2 Present address: Department of Biology, Ludwig-Maximilians-Universität Munich, D–80638 Munich, Germany. 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. 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.
Adamska I (1995) Regulation of early light-inducible protein gene expression by blue and red light in etiolated seedlings involves nuclear and plastid factors. Plant Physiol 107: 1167–1175[Abstract] Adamska I, Ohad I, Kloppstech K (1992) Synthesis of the early light-inducible protein is controlled by blue light and related to light stress. Proc Natl Acad Sci USA 89: 2610–2613 Ahmad M, Cashmore AR (1993) HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 366: 162–166[CrossRef][Medline] Ahmad M, Jarillo JA, Cashmore AR (1998a) Chimeric proteins between cry1 and cry2 Arabidopsis blue light photoreceptors indicate overlapping functions and varying protein stability. Plant Cell 10: 197–207 Ahmad M, Jarillo JA, Smirnova O, Cashmore AR (1998b) The CRY1 blue light photoreceptor of Arabidopsis interacts with phytochrome A in vitro. Mol Cell 1: 939–948[CrossRef][Web of Science][Medline] Ahmad M, Lin C, Cashmore AR (1995) Mutations throughout an Arabidopsis blue-light photoreceptor impair blue-light-responsive anthocyanin accumulation and inhibition of hypocotyl elongation. Plant J 8: 653–658[CrossRef][Web of Science][Medline] Ang LH, Chattopadhyay S, Wei N, Oyama T, Okada K, Batschauer A, Deng XW (1998) Molecular interaction between COP1 and HY5 defines a regulatory switch for light control of Arabidopsis development. Mol Cell 1: 213–222[CrossRef][Web of Science][Medline] Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55: 373–399[CrossRef][Medline] Aro EM, McCaffery S, Anderson JM (1993a) Photoinhibition and D1 protein degradation in peas acclimated to different growth irradiances. Plant Physiol 103: 835–843[Abstract] Aro EM, Virgin I, Andersson B (1993b) Photoinhibition of photosystem II: inactivation, protein damage and turnover. Biochim Biophys Acta 1143: 113–134[Medline] Benedict C, Geisler M, Trygg J, Huner N, Hurry V (2006) Consensus by democracy: using meta-analyses of microarray and genomic data to model the cold acclimation signaling pathway in Arabidopsis. Plant Physiol 141: 1219–1232 Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12: 2383–2394 Brown BA, Cloix C, Jiang GH, Kaiserli E, Herzyk P, Kliebenstein DJ, Jenkins GI (2005) A UV-B-specific signaling component orchestrates plant UV protection. Proc Natl Acad Sci USA 102: 18225–18230 Cashmore AR (2003) Cryptochromes: enabling plants and animals to determine circadian time. Cell 114: 537–543[CrossRef][Web of Science][Medline] Chattopadhyay S, Ang LH, Puente P, Deng XW, Wei N (1998) Arabidopsis bZIP protein HY5 directly interacts with light-responsive promoters in mediating light control of gene expression. Plant Cell 10: 673–683 Chen M, Chory J, Fankhauser C (2004) Light signal transduction in higher plants. Annu Rev Genet 38: 87–117[CrossRef][Web of Science][Medline] Ehrenshaft M, Bilski P, Li MY, Chignell CF, Daub ME (1999) A highly conserved sequence is a novel gene involved in de novo vitamin B6 biosynthesis. Proc Natl Acad Sci USA 96: 9374–9378 Escoubas JM, Lomas M, LaRoche J, Falkowski PG (1995) Light intensity regulation of cab gene transcription is signaled by the redox state of the plastoquinone pool. Proc Natl Acad Sci USA 92: 10237–10241 Fankhauser C, Staiger D (2002) Photoreceptors in Arabidopsis thaliana: light perception, signal transduction and entrainment of the endogenous clock. Planta 216: 1–16[CrossRef][Web of Science][Medline] Folta KM, Pontin MA, Karlin-Neumann G, Bottini R, Spalding EP (2003) Genomic and physiological studies of early cryptochrome 1 action demonstrate roles for auxin and gibberellin in the control of hypocotyl growth by blue light. Plant J 36: 203–214[CrossRef][Web of Science][Medline] Foyer CH, Allen JF (2003) Lessons from redox signaling in plants. Antioxid Redox Signal 5: 3–5[CrossRef][Web of Science][Medline] Fuglevand G, Jackson JA, Jenkins GI (1996) UV-B, UV-A, and blue light signal transduction pathways interact synergistically to regulate chalcone synthase gene expression in Arabidopsis. Plant Cell 8: 2347–2357[Abstract] Gao Y, Li J, Strickland E, Hua S, Zhao H, Chen Z, Qu L, Deng XW (2004) An Arabidopsis promoter microarray and its initial usage in the identification of HY5 binding targets in vitro. Plant Mol Biol 54: 683–699[CrossRef][Web of Science][Medline] Geisler M, Kleczkowski LA, Karpinski S (2006) A universal algorithm for genome-wide in silicio identification of biologically significant gene promoter putative cis-regulatory-elements; identification of new elements for reactive oxygen species and sucrose signaling in Arabidopsis. Plant J 45: 384–398[CrossRef][Web of Science][Medline] Goulas E, Schubert M, Kieselbach T, Kleckowski L, Gardeström P, Schröder WP, Hurry V (2006) The chloroplast lumen and stromal proteomes of Arabidopsis thaliana show differential sensitivity to short- and long-term exposure to low temperature. Plant J 47: 720–734[CrossRef][Web of Science][Medline] Guo H, Yang H, Mockler TC, Lin C (1998) Regulation of flowering time by Arabidopsis photoreceptors. Science 279: 1360–1363 Harari-Steinberg O, Ohad I, Chamovitz DA (2001) Dissection of the light signal transduction pathways regulating the two early light-induced protein genes in Arabidopsis. Plant Physiol 127: 986–997 Hardtke CS, Gohda K, Osterlund MT, Oyama T, Okada K, Deng XW (2000) HY5 stability and activity in Arabidopsis is regulated by phosphorylation in its COP1 binding domain. EMBO J 19: 4997–5006[CrossRef][Web of Science][Medline] Heddad M, Noren H, Reiser V, Dunaeva M, Andersson B, Adamska I (2006) Differential expression and localization of early light-induced proteins in Arabidopsis. Plant Physiol 142: 75–87 Holm M, Ma LG, Qu LJ, Deng XW (2002) Two interacting bZIP proteins are direct targets of COP1-mediated control of light-dependent gene expression in Arabidopsis. Genes Dev 16: 1247–1259 Huner NPA, Öquist G, Sarhan F (1998) Energy balance and acclimation to light and cold. Trends Plant Sci 3: 224–230[CrossRef][Web of Science] Hurry V, Strand A, Furbank R, Stitt M (2000) The role of inorganic phosphate in the development of freezing tolerance and the acclimatization of photosynthesis to low temperature is revealed by the pho mutants of Arabidopsis thaliana. Plant J 24: 383–396[CrossRef][Web of Science][Medline] Jain SK, Lim G (2001) Pyridoxine and pyridoxamine inhibits superoxide radicals and prevents lipid peroxidation, protein glycosylation, and (Na+ + K+)-ATPase activity reduction in high glucose-treated human erythrocytes. Free Radic Biol Med 30: 232–237[CrossRef][Web of Science][Medline] Jarillo JA, Gabrys H, Capel J, Alonso JM, Ecker JR, Cashmore AR (2001) Phototropin-related NPL1 controls chloroplast relocation induced by blue light. Nature 410: 952–954[CrossRef][Medline] Jiao Y, Yang H, Ma L, Sun N, Yu H, Liu T, Gao Y, Gu H, Chen Z, Wada M, et al (2003) A genome-wide analysis of blue-light regulation of Arabidopsis transcription factor gene expression during seedling development. Plant Physiol 133: 1480–1493 Kagawa T, Sakai T, Suetsugu N, Oikawa K, Ishiguro S, Kato T, Tabata S, Okada K, Wada M (2001) Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response. Science 291: 2138–2141 Kagawa T, Wada M (2000) Blue light-induced chloroplast relocation in Arabidopsis thaliana as analyzed by microbeam irradiance. Plant Cell Physiol 41: 84–93[Web of Science][Medline] Karpinski S, Escobar C, Karpinska B, Creissen G, Mullineaux PM (1997) Photosynthetic electron transport regulates the expression of cytosolic ascorbate peroxidase genes in Arabidopsis during excess light stress. Plant Cell 9: 627–640[Abstract] Karpinski S, Gabrys H, Mateo A, Karpinska B, Mullineaux PM (2003) Light perception in plant disease defence signalling. Curr Opin Plant Biol 6: 390–396[CrossRef][Web of Science][Medline] Karpinski S, Reynolds H, Karpinska B, Wingsle G, Creissen G, Mullineaux P (1999) Systemic signaling and acclimation in response to excess excitation energy in Arabidopsis. Science 284: 654–657 Kiedrowski S, Kawalleck P, Hahlbrock K, Somssich IE, Dangl JL (1992) Rapid activation of a novel plant defense gene is strictly dependent on the Arabidopsis RPM1 disease resistance locus. EMBO J 11: 4677–4684[Web of Science][Medline] Kimura M, Yamamoto YY, Seki M, Sakurai T, Sato M, Abe T, Yoshida S, Manabe K, Shinozaki K, Matsui M (2003) Identification of Arabidopsis genes regulated by high light-stress using cDNA microarray. Photochem Photobiol 77: 226–233[CrossRef][Web of Science][Medline] Kimura M, Yoshizumi T, Manabe K, Yamamoto YY, Matsui M (2001) Arabidopsis transcriptional regulation by light stress via hydrogen peroxide-dependent and -independent pathways. Genes Cells 6: 607–617[Abstract] Lemon WJ, Liyanarachchi S, You M (2003) A high performance test of differential gene expression for oligonucleotide arrays. Genome Biol 4: R67[CrossRef][Medline] Li J, Brader G, Palva ET (2004) The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16: 319–331 Lin C, Ahmad M, Cashmore AR (1996) Arabidopsis cryptochrome 1 is a soluble protein mediating blue light-dependent regulation of plant growth and development. Plant J 10: 893–902[CrossRef][Web of Science][Medline] Lin C, Yang H, Guo H, Mockler T, Chen J, Cashmore AR (1998) Enhancement of blue-light sensitivity of Arabidopsis seedlings by a blue light receptor cryptochrome 2. Proc Natl Acad Sci USA 95: 2686–2690 Lundmark M, Cavaco AM, Trevanion S, Hurry V (2006) Carbon partitioning and export in transgenic Arabidopsis thaliana with altered capacity for sucrose synthesis grown at low temperature: a role for metabolite transporters. Plant Cell Environ 29: 1703–1714[CrossRef][Medline] Ma L, Li J, Qu L, Hager J, Chen Z, Zhao H, Deng XW (2001) Light control of Arabidopsis development entails coordinated regulation of genome expression and cellular pathways. Plant Cell 13: 2589–2607 Mas P, Devlin PF, Panda S, Kay SA (2000) Functional interaction of phytochrome B and cryptochrome 2. Nature 408: 207–211[CrossRef][Medline] Maxwell BB, Andersson CR, Poole DS, Kay SA, Chory J (2003) HY5, circadian clock-associated 1, and a cis-element, DET1 dark response element, mediate DET1 regulation of chlorophyll a/b-binding protein 2 expression. Plant Physiol 133: 1565–1577 Neff MM, Chory J (1998) Genetic interactions between phytochrome A, phytochrome B, and cryptochrome 1 during Arabidopsis development. Plant Physiol 118: 27–35 Ohgishi M, Saji K, Okada K, Sakai T (2004) Functional analysis of each blue light receptor, cry1, cry2, phot1, and phot2, by using combinatorial multiple mutants in Arabidopsis. Proc Natl Acad Sci USA 101: 2223–2228 Oravecz A, Baumann A, Mate Z, Brezinska A, Molinier J, Oakeley EJ, Adam E, Schäfer E, Nagy F, Ulm R (2006) CONSTITUTIVELY PHOTOMORPHOGENIC1 is requited for the UV-B response in Arabidopsis. Plant Cell 18: 1975–1990 Osterlund MT, Hardtke CS, Wei N, Deng XW (2000) Targeted destabilization of HY5 during light-regulated development of Arabidopsis. Nature 405: 462–466[CrossRef][Medline] Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45 Pfannschmidt T (2003) Chloroplast redox signals: how photosynthesis controls its own genes. Trends Plant Sci 8: 33–41[CrossRef][Web of Science][Medline] Pfannschmidt T, Nilsson A, Allen JF (1999) Photosynthetic control of chloroplast gene expression. Nature 397: 625–628[CrossRef][Web of Science] Pfannschmidt T, Schutze K, Brost M, Oelmuller R (2001) A novel mechanism of nuclear photosynthesis gene regulation by redox signals from the chloroplast during photosystem stoichiometry adjustment. J Biol Chem 276: 36125–36130 Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophyll a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975: 384–394 Ramakers C, Ruijter JM, Deprez RH, Moorman AF (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339: 62–66[CrossRef][Web of Science][Medline] Richly E, Dietzmann A, Biehl A, Kurth J, Laloi C, Apel K, Salamini F, Leister D (2003) Covariations in the nuclear chloroplast transcriptome reveal a regulatory master-switch. EMBO Rep 4: 491–498[CrossRef][Web of Science][Medline] Rintamaki E, Salonen M, Suoranta UM, Carlberg I, Andersson B, Aro EM (1997) Phosphorylation of light-harvesting complex II and photosystem II core proteins shows different irradiance-dependent regulation in vivo: application of phosphothreonine antibodies to analysis of thylakoid phosphoproteins. J Biol Chem 272: 30476–30482 Rossel JB, Wilson IW, Pogson BJ (2002) Global changes in gene expression in response to high light in Arabidopsis. Plant Physiol 130: 1109–1120 Rossini S, Casazza AP, Engelmann EC, Havaux M, Jennings RC, Soave C (2006) Suppression of both ELIP1 and ELIP2 in Arabidopsis does not affect tolerance to photoinhibition and photooxidative stress. Plant Physiol 141: 1264–1273 Sakai T, Kagawa T, Kasahara M, Swartz TE, Christie JM, Briggs WR, Wada M, Okada K (2001) Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation. Proc Natl Acad Sci USA 98: 6969–6974 Schmid M, Uhlenhaut NH, Godard F, Demar M, Bressan R, Weigel D, Lohmann JU (2003) Dissection of floral induction pathways using global expression analysis. Development 130: 6001–6012 Seki M, Kamei A, Yamaguchi-Shinozaki K, Shinozaki K (2003) Molecular responses to drought, salinity and frost: common and different paths for plant protection. Curr Opin Biotechnol 14: 194–199[CrossRef][Web of Science][Medline] Shalitin D, Yang H, Mockler TC, Maymon M, Guo H, Whitelam GC, Lin C (2002) Regulation of Arabidopsis cryptochrome 2 by blue-light-dependent phosphorylation. Nature 417: 763–767[CrossRef][Medline] Shalitin D, Yu X, Maymon M, Mockler T, Lin C (2003) Blue light-dependent in vivo and in vitro phosphorylation of Arabidopsis cryptochrome 1. Plant Cell 15: 2421–2429 Tambasco-Studart M, Titiz O, Raschle T, Forster G, Amrhein N, Fitzpatrick TB (2005) Vitamin B6 biosynthesis in higher plants. Proc Natl Acad Sci USA 102: 13687–13692 Tepperman JM, Zhu T, Chang HS, Wang X, Quail PH (2001) Multiple transcription-factor genes are early targets of phytochrome A signaling. Proc Natl Acad Sci USA 98: 9437–9442 Thijs G, Marchal K, Lescot M, Rombauts S, De Moor B, Rouze P, Moreau Y (2002) A Gibbs sampling method to detect overrepresented motifs in the upstream regions of coexpressed genes. J Comput Biol 9: 447–464[CrossRef][Web of Science][Medline] Ulm R, Baumann A, Oravecz A, Mate Z, Adam E, Oakeley EJ, Schafer E, Nagy F (2004) Genome-wide analysis of gene expression reveals function of the bZIP transcription factor HY5 in the UV-B response of Arabidopsis. Proc Natl Acad Sci USA 101: 1397–1402 van Kooten O, Snel J (1990) The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth Res 25: 147–150[CrossRef][Web of Science] Vanderauwera S, Zimmermann P, Rombauts S, Vandenabeele S, Langebartels C, Gruissem W, Inze D, Van Breusegem F (2005) Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis. Plant Physiol 139: 806–821 Vener AV, Ohad I, Andersson B (1998) Protein phosphorylation and redox sensing in chloroplast thylakoids. Curr Opin Plant Biol 1: 217–223[CrossRef][Web of Science][Medline] Vidi PA, Kanwischer M, Baginsky S, Austin JR, Csucs G, Dormann P, Kessler F, Brehelin C (2006) Tocopherol cyclase (VTE1) localization and vitamin E accumulation in chloroplast plastoglobule lipoprotein particles. J Biol Chem 281: 11225–11234 Wagner U, Edwards R, Dixon DP, Mauch F (2002) Probing the diversity of the Arabidopsis glutathione S-transferase gene family. Plant Mol Biol 49: 515–532[CrossRef][Web of Science][Medline] Walters RG, Rogers JJ, Shephard F, Horton P (1999) Acclimation of Arabidopsis thaliana to the light environment: the role of photoreceptors. Planta 209: 517–527[CrossRef][Web of Science][Medline] Weston E, Thorogood K, Vinti G, Lopez-Juez E (2000) Light quantity controls leaf-cell and chloroplast development in Arabidopsis thaliana wild type and blue-light-perception mutants. Planta 211: 807–815[CrossRef][Web of Science][Medline] Yang HQ, Tang RH, Cashmore AR (2001) The signaling mechanism of Arabidopsis CRY1 involves direct interaction with COP1. Plant Cell 13: 2573–2587 Ytterberg AJ, Peltier JB, van Wijk KJ (2006) Protein profiling of plastoglobules in chloroplasts and chromoplasts: a surprising site for differential accumulation of metabolic enzymes. Plant Physiol 140: 984–997 This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
2, P < 0.05) transcriptional responses during exposure HL and BL in wild-type, cry1, and hy5 Arabidopsis backgrounds
thijs/Work/MotifSampler.html
