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Acclimation to Singlet Oxygen Stress in Chlamydomonas reinhardtii

Heidi K. Ledford, Brian L. Chin, and Krishna K. Niyogi^*

Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California 94720-3102

Received 30 June 2006/ Accepted 2 April 2007

	ABSTRACT

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In an aerobic environment, responding to oxidative cues is critical for physiological adaptation (acclimation) to changing environmental conditions. The unicellular alga Chlamydomonas reinhardtii was tested for the ability to acclimate to specific forms of oxidative stress. Acclimation was defined as the ability of a sublethal pretreatment with a reactive oxygen species to activate defense responses that subsequently enhance survival of that stress. C. reinhardtii exhibited a strong acclimation response to rose bengal, a photosensitizing dye that produces singlet oxygen. This acclimation was dependent upon photosensitization and occurred only when pretreatment was administered in the light. Shifting cells from low light to high light also enhanced resistance to singlet oxygen, suggesting an overlap in high-light and singlet oxygen response pathways. Microarray analysis of RNA levels indicated that a relatively small number of genes respond to sublethal levels of singlet oxygen. Constitutive overexpression of either of two such genes, a glutathione peroxidase gene and a glutathione S-transferase gene, was sufficient to enhance singlet oxygen resistance. Escherichia coli and Saccharomyces cerevisiae exhibit well-defined responses to reactive oxygen but did not acclimate to singlet oxygen, possibly reflecting the relative importance of singlet oxygen stress for photosynthetic organisms.

	INTRODUCTION

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Reactive oxygen species (ROS) production is an unavoidable consequence of life in an aerobic environment, and reliance upon oxygenic photosynthesis presents plants and algae with sources of ROS not generally shared by their nonphotosynthetic counterparts. Nearly any form of biotic or abiotic stress affects the chloroplast, where the photosynthetic electron transport chain brings together photosensitizing pigments, redox-active electron carriers, and oxygen generation in a polyunsaturated lipid environment. Disruptions in the balance between incoming excitation energy and terminal electron acceptors can result in ROS production and eventual cell death. High-light (HL) stress, for example, leads to increased production of singlet oxygen (¹O₂*), hydrogen peroxide, and superoxide in the chloroplast (31, 44), while hypersensitive responses to tobacco mosaic virus in tobacco result in down-regulation of the proteins necessary to repair ROS-mediated damage to photosystem II (71). Understanding how plants and algae respond to ROS and limit ROS-induced damage is therefore necessary to piece together responses to biotic and abiotic stress.

As a result of the capacity of ROS for damaging cellular constituents, including proteins, nucleic acids, and membranes (41), ROS are often cast in a purely destructive role. Evidence is emerging, however, that sublethal levels of ROS can be important signaling intermediates (4, 30), activating pathways that bolster defense responses and enhance survival of subsequent stress (11, 13, 47, 78). For example, in the yeast Saccharomyces cerevisiae, sublethal levels of hydrogen peroxide activate the YAP1 (yeast activator protein 1) transcription factor (15, 85), which then promotes expression of a number of antioxidant-related genes (35, 54), including thioredoxin (51), glutathione peroxidase (48), and gamma-glutamylcysteine synthetase (GSH1), the rate-limiting enzyme in glutathione biosynthesis (86). S. cerevisiae also acclimates to superoxide (25, 49) and lipid hydroperoxides (17), and these responses are often characterized by specificity to the form of the original stress (2, 80). GSH1, for example, is induced by both superoxide and peroxide, but loss of YAP1 abolishes peroxide induction of GSH1, while leaving superoxide induction intact (76). Acclimation responses to hydrogen peroxide and superoxide are also regulated by different response regulons in Escherichia coli (40, 78, 81). The lessons learned from E. coli and S. cerevisiae indicate that the nature of ROS signaling depends on the chemical identity of the ROS. Therefore, to understand the mechanisms by which cells sense and respond to oxidative stress, it is necessary to investigate responses to individual ROS.

Although ROS sensors in E. coli and S. cerevisiae have been characterized, the absence of obvious homologs of these sensors in algae and plants suggests that mechanisms for responding to ROS may differ in photosynthetic organisms (reviewed in reference 5). Furthermore, the abundance of photosensitizing pigments required for photosynthesis means that plants and algae may be subject to oxidative stresses, such as ¹O₂*, that are not as important for nonphotosynthetic organisms. Despite the possible importance of ¹O₂* stress responses in photosynthetic organisms, little is known about what systems may exist to counteract ¹O₂* damage. ¹O₂* is a highly reactive, excited state of oxygen that can be formed when excited triplet chlorophyll (³Chl*) in photosystem II interacts with ground-state oxygen. Environmental stress that upsets the balance between light harvesting and energy utilization lengthens the lifetime of chlorophyll (¹Chl*) (reaction 1), increasing the likelihood that ¹Chl* will undergo intersystem crossing to form ³Chl* (reaction 2). ³Chl* is longer-lived than ¹Chl* and reacts more readily with ground-state ³O₂ (reaction 3). The physical interaction between ³Chl* and oxygen produces ¹O₂* (reaction 3), liberating oxygen from the spin restriction that normally limits its reactivity with singlet-state biological molecules (39).

The three reactions are as follows: reaction 1, ¹Chl + light ¹Chl*; reaction 2, ¹Chl* ³Chl*; reaction 3, ³Chl* + ³O₂ ¹Chl + ¹O₂*.

While pigments, such as chlorophyll and protochlorophyllide, can generate ¹O₂* endogenously, exogenous photosensitizing dyes, such as rose bengal (RB), generate ¹O₂* as well (77). ¹O₂* is highly reactive and can modify lipids (36), nucleic acids (58), and proteins (14). Experiments using lipophilic photosensitizers in E. coli established that a ¹O₂* molecule could not travel more than 0.07 µm within a cell before either being quenched or reacting with another molecule (60), but recent work using a microscope capable of detecting near-infrared phosphorescence from ¹O₂* has indicated that ¹O₂* generated in the cytoplasm is capable of moving across cell membranes (75).

Despite the transience of ¹O₂*, several lines of evidence indicate that ¹O₂* can impact gene expression in photosynthetic organisms. Previous work in the single-celled alga Chlamydomonas reinhardtii established ¹O₂*-mediated regulation of a putative glutathione peroxidase gene (GPXH) (21, 23, 55). The photosynthetic proteobacterium Rhodobacter sphaeroides also induces a glutathione peroxidase in response to singlet oxygen (37), and multiple R. sphaeroides operons have been identified that appear to be under ¹O₂* control (3). Recently, work with protochlorophyllide-accumulating flu mutants in Arabidopsis thaliana has shown that ¹O₂* generated by protochlorophyllide accumulation in the chloroplast can trigger gene expression changes in the nucleus, many of which are specific to singlet oxygen and are not mimicked by treatment with hydrogen peroxide or superoxide (34, 63). ¹O₂* responses in flu mutants include growth arrest and programmed cell death, both of which are controlled by the nucleus-encoded, chloroplast-localized protein EX1 (EXECUTER 1) (83). Despite this array of physiological responses to singlet oxygen, acclimation to singlet oxygen has not yet been demonstrated in any of these systems.

To learn more about how C. reinhardtii responds to photooxidative stress, we assayed for the ability to acclimate to specific forms of ROS. We found that sublethal levels of ¹O₂* triggered a clear enhancement of defenses against ¹O₂*. Characterization of this response revealed that the abundance of transcripts of a small subset of genes was enhanced in response to ¹O₂* pretreatment. Constitutive overexpression of either of two of these genes—a glutathione peroxidase gene and a glutathione S-transferase gene—was sufficient to promote ¹O₂* resistance. The inability of S. cerevisiae and E. coli to acclimate to ¹O₂* suggests the importance of ¹O₂* responses for photosynthetic organisms.

	MATERIALS AND METHODS

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Strains and growth conditions. The wild-type C. reinhardtii strain used in this work was 4A+ (16). The pc1 y7 double mutant strain was produced by crossing CC-2471 (pc1 mt+) to CC-1174 (y7 mt–) and selecting for strains that failed to green in both the light and dark (28). The pc1 and y7 parental strains were obtained from the C. reinhardtii Stock Center.

Cells were grown photoautotrophically in minimal (HS) medium or photoheterotrophically in acetate-containing (TAP) medium under low-light (LL) conditions (50 µmol photons m^–2 s^–1) as described previously (7). Cultures were grown to mid-exponential phase (1 x 10⁶ to 2 x 10⁶ cells/ml). For LL to HL transitions, cells were shifted to 500 µmol photons m^–2 s^–1. RB (Sigma), hydrogen peroxide (EM Science), methyl viologen (Sigma), neutral red (Sigma), tert-butyl hydroperoxide (Sigma), and metronidazole (Sigma) were each dissolved in water and added directly to the growth medium immediately prior to use. Deuterium oxide, neutral red, and tert-butyl hydroperoxide assays were carried out in 100-µl volumes in 96-well trays at 50 to 60 µmol photons m^–2 s^–1. Deuterium oxide experiments were performed in TAP medium containing 95% (vol/vol) deuterium oxide (Sigma). Cells were incubated in this mix for 2 h prior to RB treatment.

Experiments with E. coli were performed using strain DH5 in Luria broth at 37°C. Experiments with S. cerevisiae were performed at 30°C in yeast extract-peptone-dextrose (YPD) medium using strain YPH500 (73). Maximum pretreatment concentrations used were the highest concentrations of RB that did not result in cell death.

Tocopherol and pigment analysis. Wild-type cells were grown photoautotrophically in 100-ml cultures under LL. RB treatments were administered as described above, and 2-ml samples were taken for acetone extraction and high-performance liquid chromatography determination of tocopherol and pigment content as described previously (6).

RNA isolation. Samples were harvested by centrifugation (3,200 x g, 4°C, 3 min) and then resuspended in 0.1 volume of H₂O at 4°C. An equal volume of 2x lysis buffer (0.6 M NaCl, 10 mM EDTA, 100 mM Tris HCl [pH 8.0], 4% [wt/vol] sodium dodecyl sulfate [SDS]) was added to the cell suspension, which was subsequently incubated at 65°C for 5 min. Then, 0.132 volume of 2 M KCl was added, and the samples were incubated on ice for 15 min. Samples were then centrifuged at 12,000 x g for 10 min at 4°C. Supernatants were extracted twice with phenol-chloroform and once with chloroform before precipitating RNA overnight using 0.33 volume of 8 M LiCl at 4°C. This was followed by a final ethanol precipitation. RNA quality was assessed using the ratio of absorbance at 260 nm and 280 nm and by ethidium bromide staining following gel electrophoresis. RNA for both microarrays and RNA gel blot analysis was isolated in this manner.

Microarray experimental design. Cultures (100 ml) of C. reinhardtii 4A+ were grown photoautotrophically at 50 µmol photons m^–2 s^–1 until they reached a density of 1.5 x 10⁶ cells/ml. "Pretreated" samples were treated with 2 µM RB, whereas "unpretreated" cultures received a mock inoculation with an equal volume of water. Cells were incubated for 2 h before RNA was harvested.

Microarray slides were printed as part of the C. reinhardtii Genome Project (46, 72). Fragments corresponding to the last 400 base pairs at the 3' ends of 2,761 C. reinhardtii cDNAs were amplified and spotted onto polyamine-coated slides (Corning, Acton, MA). Each slide contained four replicate spots arrayed in four distinct grids. A total of four slides were used, encompassing two biological replicates, each with a dye-switch control.

Microarray probe labeling. Reverse transcription of RNA samples was carried out at 42°C for 2 h in the presence of deoxynucleoside triphosphates containing a 1:1 ratio of amino-allyl dUTP to dTTP. Samples were treated with EDTA to stop the reaction and with NaOH to destroy RNA. After neutralizing the sample with HCl, cDNA was purified using a Microcon 30 column, dried, and resuspended in 0.1 M Na₂HCO₃ buffer (pH 9.0). RNA samples were labeled using Post-Labeling Reactive Dye Packs from Amersham Biosciences (Amersham, Little Chalfont, Buckinghamshire, United Kingdom). Probes were purified individually using QIAquick columns (QIAGEN, Valencia, CA) and then combined.

Microarray hybridization conditions. Slides were blocked in a succinic anhydride-sodium borate solution for 20 min and then rehydrated in a boiling water bath for 1 min. Slides were then rinsed in ethanol and dried by brief centrifugation. Prehybridization was carried out for 20 min at 50°C in a solution containing 3.5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% (wt/vol) SDS, and 10 mg/ml bovine serum albumin. After prehybridization, slides were rinsed with water and then with isopropanol and dried. For hybridization, a 2x hybridization buffer containing 6x SSC, 0.2% (wt/vol) SDS, 0.1 mg/ml poly(dA), and 0.1 mg/ml yeast tRNA was prepared and mixed with an equal volume of labeled cDNA. Slides were hybridized overnight in a 50°C water bath. The next day, slides were washed once in 2x SSC, 0.03% (wt/vol) SDS, once in 1x SSC, and once in 0.05x SSC, each for 5 min. Slides were then dried and stored until scanning.

Microarray image analysis. Slides were scanned on an ArrayWoRx Biochip Reader and quantitated using the SoftWoRx Tracker Microarray program, both from Applied Precision, LLC (Issaquah, WA). Each represented gene had to have a valid data point from each biological replicate to be considered further. Abnormal spots were flagged manually and excluded from further analysis, as was any spot in which the mean spot intensity did not exceed two times the median background intensity, or in which the signal-to-noise ratio value was less than 1. Spots that were >20% saturated were also excluded.

Data analysis. Each slide contained four replicates of each spot arrayed in separate grids. For data normalization, each grid (containing a single set of 2,761 C. reinhardtii cDNA PCR products) was normalized individually. Preliminary analysis indicated that considerably fewer than 5% of the data points showed a greater than twofold change, suggesting that scaling could be an appropriate method for normalization of this data set. For comparison, data were also normalized manually in Excel (Microsoft). The median background intensity was subtracted from the mean spot intensity in each channel. A regression line was fit to a Cy3 versus Cy5 plot, and Cy3 values were divided by the slope of the line. Tailing was often observed at high and low intensity values. To account for this tailing, linear scaling and intensity-dependent normalization were performed using the SNOMAD program with a span of 0.7 and a trim of 0.1 (10). Data generated by both normalization methods were nearly identical. The data presented in Table 1 are ratios calculated from SNOMAD-normalized data.

RNA gel blot analysis. Primers were designed based upon the expressed sequence tag (EST) contig assembly sequences (72). The primers used to amplify GSTS1 were 5'-TTACGACTTCCTCCGCACTC-3' and 5'-CGGGACCAGACCTGTTTCTTG-3'. The primers used to amplify PHC8 were 5'-CAGTCGCCTACCACAATTCAC-3' and 5'-TGGCCTCATCTTCTCACCTTC-3'. Primers for amplification of a portion of contig number 20021010.5327 (referred to as 5327 hereafter) were 5'-GCCAGACTTGTTGTCTTATTACCAT-3' and 5'-CTGTATTTGCTGTGTAAGGGTTTG-3'. The primers used to amplify GSTS2 were designed based on the contig 20021010.3547. GSTS2 primer sequences were 5'-AAGGCCTACTACCAGGACAAGAC-3' and 5'-CTGTAAACCAAACGACTTCAAGG-3'. PCR products were cloned into the pGEM-T Easy vector (Promega, Madison, WI). GPXH probes were generated from vectors described by Leisinger et al. (55, 56). The APX1 probe was described by Ledford et al. (53). To generate RNA probes, vector inserts were transcribed in the antisense direction in the presence of digoxigenin-labeled dUTP (Roche Molecular Biochemicals, Germany).

RNA from C. reinhardtii cells was prepared as described above, and 5 µg of RNA per sample were fractionated using denaturing gel electrophoresis (67). Blots were hybridized in DIG-EasyHyb solution (Roche Molecular Biochemicals, Germany) at 67°C, and high-stringency washes were carried out at 68°C using 0.2x SSC, 0.1% (wt/vol) SDS.

Overexpression of GPXH and GSTS1. PSAD flanking sequences were used to drive constitutive overexpression of GPXH and GSTS1 (24). GPXH cDNA was amplified from the vector described by Leisinger et al. (56), using primers designed to engineer an NdeI site at the beginning and an EcoRI site at the end. The GPXH_NdeI forward primer sequence was 5'-TCACAACAAGCCCATATGGCGAACCCCGAGTTTTACG-3', and the GPXH_EcoRI reverse primer sequence was 5'-CAGCTGCTGCCAGAATTCTTAGTTACGCGTTC-3' (restriction sites are underlined). Similarly, GSTS1 was amplified from genomic DNA using the following primers: GSTS1_NdeI 5'-TCACAACAAGCCCATATGGCCCCCAAGCTGTA-3' and GSTS1_EcoRI 5'-CAGCTGCTGCCAGAATTCTTACGCGTCTGGCC-3'. PCR products were digested with EcoRI and NdeI and cloned into the pSL18 vector containing PSAD 5' and 3' untranslated regions as well as a paromomycin-selectable marker (24, 64).

Pro_PSAD:GPXH and Pro_PSAD:GSTS1 were transformed into C. reinhardtii 4A+ by the method of Dent et al. (16). Transformants were selected on TAP plates containing 10 µg/ml paromomycin (Sigma).

Microarray data accession numbers. Raw microarray data have been deposited into the National Center for Biotechnology Information Gene Expression Omnibus database at http://www.ncbi.nlm.nih.gov/geo under series accession number GSE4681 [NCBI GEO] .

	RESULTS

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C. reinhardtii exhibits a robust acclimation response to ¹O₂*. To learn more about how photosynthetic organisms respond to photooxidative stress, the model alga, C. reinhardtii, was tested for the ability to acclimate to specific ROS. Acclimation was scored as the ability of pretreatment with a sublethal level of a ROS-generating compound to induce defense responses that enhanced survival of a subsequent challenge with higher concentrations of ROS (Fig. 1A). We tested hydrogen peroxide, tert-butyl hydroperoxide (an organic peroxide capable of triggering lipid peroxidation chain reactions), metronidazole (a compound which generates superoxide only in the chloroplast) (70), and RB (a xanthene dye that produces ¹O₂* upon excitation by light) for the ability to induce an acclimation response. The response to RB was the most dramatic, with a significant proportion of pretreated cells surviving concentrations that killed all unpretreated cells (Fig. 1B and Fig. 2A, leftmost photo).

View larger version (31K): [in this window] [in a new window]   FIG. 1. C. reinhardtii acclimates to singlet oxygen. (A) Schematic of acclimation assays. C. reinhardtii was assayed for an ability to acclimate to oxidative stress following pretreatment with sublethal levels of that stress. Cells were pretreated and challenged with reactive oxygen species in liquid cultures and then plated on agar medium to assay survival. Acclimation was defined as the ability of pretreated cells to survive what would otherwise be a lethal challenge level. (B) Assay for acclimation to RB. Wild-type cells were grown photoautotrophically and pretreated in HS liquid medium for 2 h with 2 µM RB and then challenged for 1 h using the indicated concentrations. Pretreatment and challenge were carried out in 100-ml cultures at 50 µmol photons m–2 s–1. Aliquots were serially diluted and plated on TAP medium, and colonies were counted to assay the viability of the original cultures.

View larger version (29K): [in this window] [in a new window]   FIG. 2. Light and solvent dependency of responses to RB. (A) Light dependency of acclimation to RB. Cells were pretreated and challenged in the light (50 µmol photons m–2 s–1; indicated by a sun) or the dark (indicated by a moon). Pretreatment lasted 2 h, and challenge levels lasted 1 h. Cells were grown and treated in TAP liquid medium, and a 3-µl aliquot was spotted onto TAP plates as a viability assay. (B) Solvent dependency of RB toxicity. Cells were treated with the indicated concentrations of RB for 1 h either in normal aqueous TAP medium (H2O) or in TAP medium containing 95% (vol/vol) deuterium oxide (D2O).

The lifetime of ¹O₂* is approximately 10 times longer in deuterium oxide than it is in H₂O (59). Therefore, if RB toxicity is dependent upon ¹O₂*, then toxicity should be enhanced in deuterium oxide. As shown in Fig. 2B, 1 µM RB was lethal to cells treated with RB in deuterium oxide, whereas the viability of cells treated with RB in water was unaffected at that concentration.

Acclimation to ¹O₂* is rapidly induced and transient. Pretreatment times and concentrations were varied to determine how quickly acclimation is induced (Fig. 3A). Acclimation could be triggered by as little as 0.25 µM RB, and the effect increased with longer incubation times and higher concentrations of RB. A small degree of acclimation was evident with only 30 min of pretreatment, and the degree of ¹O₂* resistance increased with longer pretreatment time.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Acclimation to 1O2* is rapid and transient. (A) Cells were grown photoautotrophically to mid-exponential phase, then pretreated with various RB concentrations and durations (duration of pretreatment is indicated below each photo), and challenged for 1 h at the concentrations shown. Aliquots were spotted onto TAP agar medium to assay viability. (B) Transience of acclimation to 1O2*. Cells were grown photoautotrophically to mid-exponential phase, pretreated (+) with 2 µM RB for 2 h, and then challenged with 8 µM RB for 1 h. A 3-µl aliquot from each sample was spotted onto TAP agar medium as an assay of viability. The remaining culture from the sample that was pretreated and challenged was washed with fresh HS medium and incubated for 24 h, at which time the pretreatment and challenge were repeated. Aliquots were again spotted onto TAP agar medium, shown in the right photo.

Acclimation is specific to ¹O₂*_. To explore possible overlap between responses to different ROS, we then tested the ability of ¹O₂* pretreatment to induce acclimation to other ROS-generating compounds, including hydrogen peroxide, methyl viologen, metronidazole, tert-butyl hydroperoxide, and neutral red (another photosensitizing dye). The only evidence of cross-tolerance as the result of ¹O₂* pretreatment was observed in the case of neutral red (Fig. 4A), which has been shown to produce ¹O₂* in treated C. reinhardtii thylakoids (23). Interestingly, pretreatment with RB increased methyl viologen sensitivity but had no impact on sensitivity to metronidazole. This could be related to differences in chemical structure or sites of action between these two compounds (70). The experiment was also reversed, this time pretreating with neutral red, hydrogen peroxide, methyl viologen, metronidazole, and tert-butyl hydroperoxide, and challenging with RB. Only the neutral red pretreatment was able to enhance resistance to RB (data not shown). These results suggest that the acclimation occurs specifically in response to ¹O₂* and activates defenses that are specific to protection against ¹O₂*-mediated damage.

View larger version (21K): [in this window] [in a new window]   FIG. 4. Cross-acclimation between 1O2* and other sources of oxidative stress. (A) Cross-acclimation between 1O2* and other ROS. Cells were grown photoautotrophically, pretreated (+) with 2 µM RB for 2 h at 50 µmol photons m–2 s–1, and then challenged for 1 h at the same light intensity with either RB (0, 6, 8, 10, and 12 µM), neutral red (NR; 0, 4, 6, 8, 10 µM), tert-butyl hydroperoxide (tBOOH; 0, 0.25, 0.5, 0.75, and 1 mM), metronidazole (MZ; 0, 2, 3, 4, 6 mM), and methyl viologen (MeV; 0, 0.5, 1, 1.5, and 2 µM). (B) Cross-acclimation between 1O2* and HL. Cultures were grown photoautotrophically at 50 µmol photons m–2 s–1 (LL) and then pretreated by shifting to 500 µmol photons m–2 s–1 (HL) for 1 h prior to challenging with the indicated concentrations of RB for 5 h at LL.

Acclimation to ¹O₂* stress does not alter the composition or content of carotenoids or vitamin E. Several small-molecule antioxidants are capable of quenching ¹O₂* or scavenging ¹O₂*-produced damage products (69). Included in this group of antioxidants are carotenoids and tocopherols (vitamin E). In C. reinhardtii, it has previously been shown that alterations in carotenoid composition can affect RB sensitivity (6, 7), but there were no changes in ß-carotene, lutein, or the xanthophyll cycle pigments zeaxanthin, antheraxanthin, and violaxanthin during the 2-hour pretreatment or the 1-hour challenge (Fig. 5). Similarly, no changes in vitamin E content or composition were observed (Fig. 5). Based on these data, changes in the abundance or composition of carotenoids and vitamin E do not account for the increased resistance to ¹O₂* seen following pretreatment.

View larger version (27K): [in this window] [in a new window]   FIG. 5. Acclimation does not involve changes in cellular carotenoid or -tocopherol content or composition. Photoautotrophically grown cells were pretreated with 2 µM RB for 2 h at 50 µmol photons m–2 s–1 and then challenged for 1 h with 12 µM RB. Error bars represent standard errors from four biological replicates. Gray shaded bars, cells not pretreated; white bars, cells not pretreated but challenged; speckled bars, pretreated cells; striped bars, pretreated and challenged cells. The xanthophyll cycle pool is the sum of violaxanthin, antheraxanthin, and zeaxanthin. ß-car, ß-carotene; -toc, -tocopherol; Chl a, chlorophyll a.

Although many genes encoding proteins with possible roles in antioxidant metabolism were included on these partial-genome arrays (53), only a small number of genes changed expression in response to the sublethal pretreatment with RB (Fig. 6A). Each of these genes is listed in Table 1. Among those genes that increased expression were a glutathione peroxidase (GPXH) and a glutathione S-transferase (GSTS1). A cytosolic thioredoxin (TRXH) that has been shown to play a role in resistance to DNA alkylating agents (68) also increased expression, as did a gene encoding a predicted protein with 40% sequence identity to pherophorins from Volvox carteri (38) (PHC8). In addition, a gene with no sequence similarity to genes of known function (5327) increased expression. Among those genes that decreased expression during pretreatment were two genes related to the carbon-concentrating mechanism, the periplasmic carbonic anhydrase (CAH1) and a chloroplast envelope carrier protein (CCP1), both of which are induced in response to low CO₂ concentrations (33, 45, 65).

View larger version (56K): [in this window] [in a new window]   FIG. 6. Pretreatment with 1O2* causes changes in gene expression. (A) Volcano plot of microarray results comparing pretreated cells with unpretreated cells. Log2-transformed gene expression ratios are plotted on the x axis against log10-transformed P values (log10pval). Genes with statistically different expression levels (P value of 1 x 10–4) and changes greater than 1.5-fold are considered to be induced, and genes with statistically different expression levels and changes less than –1.5-fold are considered to be repressed. These genes fall within the boxes in the upper right and upper left corners of the graph, respectively. (B) RNA gel blot confirmation of microarray results. Cells were grown in TAP medium at either 50 µmol photons m–2 s–1 ("light") or in the dark. Pretreatment was 2 µM RB for 2 h, and challenge was 10 µM RB for 1 h. Five micrograms of RNA was loaded in each lane, and a methylene blue stain of rRNA was used as a loading control. Blots shown here are representative of two biological replicates. The levels of GPXH (glutathione peroxidase 1), GSTS1 (glutathione S-transferase 1), GSTS2 (glutathione S-transferase 2), PHC8 (pherophorin C8), and APX1 (ascorbate peroxidase) genes are shown. 5327 refers to the EST contig 20021010.5327.

Endogenous photosensitizers also induce GPXH and GSTS1 expression. Although RB appeared to be affecting cell physiology through the production of ¹O₂*, we wanted to know whether these same changes would occur in response to an endogenous source of ¹O₂*. To that end, we evaluated gene expression changes in a chlorophyll biosynthesis mutant. In C. reinhardtii, there is both a light-dependent and a light-independent pathway for the conversion of protochlorophyllide to chlorophyllide (28). The pc1 y7 double mutant is blocked in both pathways, leading to the accumulation of protochlorophyllide and inability to grow under even LL conditions (57). In the presence of light, accumulated protochlorophyllide can act as an endogenous photosensitizer and generate ¹O₂* within the chloroplast (63). RNA was isolated from pc1 y7 and wild-type cells following a shift from the dark to LL, and RNA gel blot analysis was used to evaluate gene expression changes immediately following this shift. Transcript abundance of both GPXH and GSTS1 increased to higher levels in the pc1 y7 mutant relative to the wild type in response to the transfer from dark to LL (Fig. 7A). GPXH exhibited a more rapid response, with transcript abundance increasing after only 30 min in the light, whereas GSTS1 expression exhibited a slower response, increasing after 3 h.

View larger version (38K): [in this window] [in a new window]   FIG. 7. Impact of endogenous photosensitizers on GPXH and GSTS1 expression. (A) RNA gel blot analysis of pc1 y7 cells transferred from dark to LL. The protochlorophyllide-accumulating mutant pc1 y7 and the wild type (WT) were grown in TAP medium in the dark and then shifted to 50 µmol photons m–2 s–1 (LL). RNA samples were taken at the indicated time points, and 5 µg of total RNA was loaded in each lane. Methylene blue staining of rRNA was used as a loading control, and the mutant and wild-type blots were hybridized together to the same probe. Both blots are representative of two biological replicates. (B) RNA gel blot analysis of wild type transferred from LL to HL. Wild-type cells were grown photoautotrophically in LL and then shifted to 500 µmol photons m–2 s–1. Total RNA was extracted at the indicated times, and 5 µg of total RNA was run in each lane. Methylene blue staining of rRNA was used as a loading control, and both blots are representative of two biological replicates.

Specificity of ¹O₂*-induced gene expression changes. To determine whether other ROS affect expression of the ¹O₂*-responsive genes, gene expression changes in response to hydrogen peroxide, metronidazole, and tert-butyl hydroperoxide were monitored. As previously reported (55, 56), GPXH expression did not respond as strongly or as quickly to hydrogen peroxide or the superoxide generators metronidazole and methyl viologen but did respond slowly to tert-butyl hydroperoxide, an organic peroxide (Fig. 8). All other ¹O₂*-induced genes were also induced by these additional ROS. Interestingly, GSTS2 and APX1, which did not increase expression in response to ¹O₂* in heterotrophically grown cells (Fig. 6), did respond to ¹O₂* in photoautotrophically grown cells (Fig. 8). CAH1, which decreased expression in response to ¹O₂* in the microarray analysis (Table 1), also decreased expression in response to all other ROS tested (Fig. 8).

View larger version (42K): [in this window] [in a new window]   FIG. 8. Oxidative stress-induced changes in gene expression. Cells were grown photoautotrophically at 50 µmol photons m–2 s–1 and treated with the indicated compounds (tBOOH, tert-butyl hydroperoxide; MZ, metronidazole). Concentrations used did not result in cell death during 12 h of treatment. Five micrograms of total RNA was loaded in each lane, and a methylene blue stain of the blot was used as an rRNA loading control. Blots shown here are representative of two biological replicates.

View larger version (34K): [in this window] [in a new window]   FIG. 9. Constitutive overexpression of GPXH and GSTS1 confers resistance to 1O2*. (A) RNA gel blot analysis of ProPSAD:GPXH transformants. RNA was extracted from heterotrophically grown paromomycin-resistant transformants containing the ProPSAD:GPXH overexpression construct. Five micrograms of RNA was loaded in each lane, and methylene blue staining of the blot was used as a loading control. "pSL18" refers to transformants that contain an empty vector control. (B) RNA gel blot analysis of ProPSAD:GSTS1 transformants. RNA was extracted from heterotrophically grown paromomycin-resistant transformants containing the ProPSAD:GSTS1 overexpression construct. Five micrograms of RNA was loaded in each lane, and methylene blue staining of the blot was used as a loading control. "pSL18" refers to transformants that contain an empty vector control. (C) Phenotypes of ProPSAD:GPXH and ProPSAD:GSTS1. GPXH- and GSTS1-overexpressing transformants were grown in TAP liquid cultures, then plated onto TAP plates containing either 1.5 µM RB or 4 µM neutral red (NR), and grown at 100 µmol photons m–2 s–1. pSL18A1 is a control transformant containing the empty vector.

E. coli and S. cerevisiae do not acclimate to ¹O₂* stress. There are large mutant collections for E. coli and S. cerevisiae, two model organisms in which oxidative stress responses have been extensively investigated. In hopes of taking advantage of the tools available in these systems, E. coli and S. cerevisiae were tested for the ability to acclimate to RB-induced ¹O₂* stress. Concentrations of RB were established for both E. coli and S. cerevisiae that would be sublethal (for pretreatments) over different periods of exposure ranging from 15 min to 5 h. No condition was found that enhanced survival after challenge, and instead the pretreatment often had an additive, deleterious effect when followed by a challenge (Fig. 10).

View larger version (27K): [in this window] [in a new window]   FIG. 10. Screens for acclimation to 1O2* in E. coli and S. cerevisiae. E. coli and S. cerevisiae cultures were pretreated with the indicated RB concentrations for 2 h, then challenged for 1 h, and plated.

	DISCUSSION

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View larger version (16K): [in this window] [in a new window]   FIG. 11. Model of acclimation to 1O2* in C. reinhardtii. Pchlide, protochlorophyllide; LOOH, lipid hydroperoxide.

The ability of HL to induce acclimation to ¹O₂* stress demonstrates an intriguing overlap between ¹O₂* responses and HL exposure (Fig. 4B) and suggests that endogenous ¹O₂* (produced by excited chlorophyll) can induce acclimation to exogenous ¹O₂* (produced by RB) (Fig. 11). In its natural environment, C. reinhardtii would regularly experience photon flux densities equivalent to our HL treatment, which represents 25% of full sunlight, and rapid fluctuations in light intensity similar to the 10-fold increase used in our experiments occur frequently in nature. A number of ROS are produced in response to HL, including hydrogen peroxide, superoxide, and ¹O₂* (29, 31, 44). The fact that methyl viologen, metronidazole, hydrogen peroxide, and tert-butyl hydroperoxide did not induce acclimation to ¹O₂* (Fig. 4A) implies that the HL-generated signal is likely mediated by ¹O₂* rather than hydrogen peroxide, lipid peroxides, or superoxide. However, because tert-butyl hydroperoxide is a shorter molecule than naturally occurring lipids, a role for lipid peroxides cannot be completely ruled out.

Changes in gene expression, but not carotenoid or vitamin E composition, occur during acclimation. Carotenoids are efficient ¹O₂* quenchers, and they increase ¹O₂* resistance when overexpressed in E. coli (79). A C. reinhardtii double mutant, npq1 lor1 mutant that is unable to synthesize lutein and zeaxanthin is also more sensitive than the wild type to ¹O₂* (7). Furthermore, creating a triple mutant containing npq1, lor1, and npq2, which causes cells to accumulate zeaxanthin, restores RB tolerance to wild-type levels (6). Tocopherols (vitamin E) also play a role in ¹O₂* defense, and the tocopherol-deficient vte1 mutant in A. thaliana accumulates more lipid peroxides in response to ¹O₂* than the wild type (43), but despite the potential for carotenoids and tocopherols to protect against ¹O₂*, changes in carotenoid and tocopherol composition or content did not accompany acclimation to ¹O₂* in C. reinhardtii (Fig. 5). Glutathione can also be an important component of antioxidant defenses against ¹O₂* by providing reducing power for lipid peroxide scavenging enzymes, but previous work has established that neither glutathione content nor glutathione redox state is altered by RB treatment under conditions similar to those used in this work (1 µM RB for 20 to 120 min at 120 µmol photons m^–2 s^–1) (22).

Instead, acclimation to ¹O₂* was associated with changes in nuclear gene expression. Microarray experiments using the v1.0 C. reinhardtii cDNA arrays (46, 72) detected only 14 genes that changed expression in response to pretreatment with ¹O₂* (Fig. 6A and Table 1). Six of the 14 genes (GPXH, GSTS1, PHC8, 5327, CAH1, and THI4a) were also tested by RNA gel blot analysis (Fig. 6B and 8; also data not shown), and the changes in gene expression were confirmed in each case. The arrays used in our analysis cover approximately 20% of the genome. Extrapolating from these results to the full genome yields an estimated 70 ¹O₂*-regulated genes in the C. reinhardtii genome.

Gene expression changes in response to the pretreatment were light dependent, indicating that transcript abundance changed in response to ¹O₂*, and not merely in reaction to the presence of a xenobiotic compound, such as RB (Fig. 6B). This was particularly interesting in the case of GSTS1. No longer relegated only to the role of xenobiotic detoxification, glutathione S-transferases are now known to be a diverse group of enzymes responsible for detoxifying endogenous compounds, including lipid peroxides (1, 74). Some play a direct role in signaling (1). In mammalian systems, prostaglandin H synthase-2, also a member of the sigma class of glutathione S-transferases, is induced by ROS (18). Activation of GSTS1 by RB and endogenous photosensitizers only in the light suggests that transcript abundance of this gene is regulated by oxidative stress rather than the mere presence of foreign chemicals (Fig. 6B and 7). The signal that triggers enhanced GSTS1 expression was not specific to ¹O₂*, however, and induction of both GSTS1 and GSTS2 occurred in response to each ROS tested (Fig. 8). ¹O₂* induction of APX1 and GSTS2 was more complicated, occurring only in photoautotrophically grown cultures (Fig. 6B and 8).

Decreased expression of the periplasmic carbonic anhydrase gene, CAH1, was also observed in response to each of the ROS tested (Fig. 8). CAH1 transcript levels respond rapidly to changes in CO₂, and mRNA abundance decreases within an hour after the start of CO₂ supplementation (32). The signal that triggers these changes in C. reinhardtii is as yet unknown, and the effect of ¹O₂* on CAH1 and CCP1 expression might indicate some cross talk between oxidative stress and regulation of the carbon-concentrating mechanism. In the marine diatom Phaeodactylum tricornutum, increases in cyclic AMP (cAMP) have been suggested to repress transcription of a chloroplastic carbonic anhydrase gene (42). Interestingly, a sequence motif similar to the mammalian cAMP response element has been identified within a region of the GPXH promoter that is responsible for increased transcription of this gene in response to ¹O₂* in C. reinhardtii (55).

Of all the genes that changed expression in response to ¹O₂*, only GPXH showed a stronger, more rapid response to ¹O₂* than to the other ROS tested (Fig. 8). This result confirms previously published work showing that GPXH is induced by photosensitizing dyes and organic hydroperoxides, but not by the superoxide-generating herbicides metronidazole and methyl viologen (55). Because GPXH and GSTS1 encode proteins with potential antioxidant function, it was possible that changes in expression of these genes could affect ¹O₂* resistance in C. reinhardtii. Overexpression of either GPXH or GSTS1 was sufficient to enhance ¹O₂* resistance (Fig. 9). However, pretreatment with other ROS also enhance GSTS1 expression (Fig. 8) without increasing ¹O₂* resistance (data not shown), suggesting that transient increases in GSTS1 transcript cannot be sufficient to induce ¹O₂* resistance and implying an additional level of regulation. This added layer of regulation could be at the level of translation, and singlet oxygen has been previously shown to affect translation elongation of the D1 protein in Synechocystis sp. strain PCC 6803 (61).

How do GPXH and GSTS1 enhance resistance to ¹O₂*? Sequence alignments with glutathione peroxidases from other organisms showed that GPXH exhibits features of phospholipid hydroperoxide glutathione peroxidases (data not shown) (5). Given that some glutathione S-transferases also function as lipid peroxidases, the simplest explanation is that overexpressing GPXH and GSTS1 protects cells from ¹O₂* by enhancing lipid peroxidase activity, but other possible functions for these two genes certainly have not been ruled out. For example, both glutathione peroxidases and glutathione S-transferases have been shown to play direct signaling roles as well (15, 84). Biochemical characterization of GPXH and GSTS1, as well as loss-of-function mutants or RNA interference lines, would be useful to determine the functions of these two genes during ¹O₂* acclimation.

Model of acclimation to ¹O₂*. Overall, this study has allowed us to derive a model of ¹O₂* acclimation (Fig. 11) in which ¹O₂* from exogenous dyes, such as RB (Fig. 1 and 2), or endogenous pigments, such as chlorophyll or protochlorophyllide (Fig. 7), activates a signal transduction pathway that increases expression of GPXH, GSTS1, as well as other genes (Table 1). How the ¹O₂* signal is perceived and converted to enhanced gene expression remains a mystery. There are three obvious possibilities: ¹O₂* could itself directly modify a protein sensor; a by-product of ¹O₂* damage, such as a lipid peroxide or protein peroxide, could interact with the sensor protein; or ¹O₂* could activate a sensor by perturbing the redox state of the cell (Fig. 11). At present, there is no definitive evidence for or against any of these possibilities. Regulation of GPXH expression by ¹O₂* has been mapped to two regions of the promoter (55), one of which contains a putative cAMP-response element that is also found in predicted introns of both GSTS1 and GSTS2 (data not shown). Future work evaluating the role of this element in ¹O₂* signaling could be valuable for piecing together how this short-lived signal is sensed.

HL-induced acclimation to ¹O₂* stress demonstrates the presence of a chloroplast-to-nucleus retrograde signaling pathway capable of activating the acclimation response. Studies of ¹O₂* responses in the flu mutant of A. thaliana have also demonstrated a ¹O₂*-activated retrograde signaling pathway (63, 83). In A. thaliana, this pathway is mediated by EX1. flu ex1 double mutants produce as much ¹O₂* as flu single mutants but do not experience either growth arrest or cell death in response to light/dark cycles (83). This suggests a signaling rather than antioxidant role for the chloroplast-localized EX1 and also indicates that, in the flu mutant, cell death in response to ¹O₂* is genetically programmed rather than the direct product of oxidative damage. Whether this response to ¹O₂* is conserved in C. reinhardtii is currently unknown, but there is a gene with sequence similarity to EX1 in the current release of the C. reinhardtii genome. Future work will address the role of this gene in C. reinhardtii.

This work expands our knowledge of biological responses to ¹O₂* and raises questions about the nature of the sensing and signaling pathways involved in acclimation to ¹O₂*. The physiological and molecular characterization described here opens the door for genetic approaches to dissect these pathways. The strong acclimation to ¹O₂* exhibited by C. reinhardtii makes it an ideal model photosynthetic eukaryote in which to pursue studies of ¹O₂*.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge Chung-Soon Im and Arthur Grossman for assistance with microarray analyses, Trevor Starr for assistance with S. cerevisiae experiments, and Setsuko Wakao for critical reading of the manuscript.

This work was supported by grants from the National Institutes of Health (GM071908) and the University of California Toxic Substances Research and Teaching Program (03T-1) to K.K.N. H.K.L. was supported in part by a National Institutes of Health Predoctoral Genetics training grant (T32-GM07127).

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, Berkeley, CA 94720-3102. Phone: (510) 643-6602. Fax: (510) 642-4995. E-mail: niyogi{at}nature.berkeley.edu

Published ahead of print on 13 April 2007.

Present address: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142.

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