JXB Advance Access originally published online on January 22, 2008
Journal of Experimental Botany 2008 59(2):121-133; doi:10.1093/jxb/erm289
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FOCUS PAPER |
Impact of chloroplastic- and extracellular-sourced ROS on high light-responsive gene expression in Arabidopsis
1Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK
2Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes, CNRS, Place Viala, 34060 Montpellier cedex 1, France
3John Innes Centre, Colney, Norwich NR4 7UH, UK
4Department of Botany, Stockholm University, 106 91 Stockholm, Sweden
5ARC Centre of Excellence in Plant Energy Biology, School of Biochemistry and Molecular Biology, The Australian National University, Canberra, ACT 0200, Australia
To whom correspondence should be addressed. E-mail: mullin{at}essex.ac.uk
Received 14 June 2007; Revised 2 October 2007 Accepted 5 November 2007
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Abstract |
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The expression of 28 high light (HL)-responsive genes of Arabidopsis was analysed in response to environmental and physiological factors known to influence the expression of the HL-responsive gene, ASCORBATE PEROXIDASE2 (APX2). Most (81%) of the HL-responsive genes, including APX2, required photosynthetic electron transport for their expression, and were responsive to abscisic acid (ABA; 68%), strengthening the impression that these two signals are crucial in the expression of HL-responsive genes. Further, from the use of mutants altered in reactive oxygen species (ROS) metabolism, it was shown that 61% of these genes, including APX2, may be responsive to chloroplast-sourced ROS. In contrast, apoplastic/plasma membrane-sourced H2O2, in part directed by the respiratory burst NADPH oxidases AtrbohD and AtrbohF, was shown to be important only for APX2 expression. APX2 expression in leaves is limited to bundle sheath parenchyma; however, for the other genes in this study, information on their tissue specificity of expression is sparse. An analysis of expression in petioles, enriched for bundle sheath tissue compared with distal leaf blade, in HL and control leaves showed that 25% of them had >10-fold higher expression in the petiole than in the leaf blade. However, this did not mean that these petiole expression genes followed a pattern of regulation observed for APX2.
Key words: Arabidopsis, chloroplast, excess light, gene expression, plasma membrane, reactive oxygen species, signalling
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Introduction |
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Exposure of plants to light levels considerably in excess of those required to saturate carbon metabolism can result in increased rates of electron transport in the absence of any increase in CO2 assimilation (Fryer et al., 1997; Ort and Baker, 2002). Under such conditions, molecular oxygen (O2) can be used as an alternative electron acceptor to CO2 in two ways (Asada, 1999; Ort and Baker, 2002). First, O2 can be photoreduced by photosystem I (PSI) to produce superoxide anion radicals (O2·–), which are rapidly converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD; Asada, 1999). Secondly, photorespiration can also consume photosynthetic reducing equivalents and indirectly produce H2O2 (Asada, 1999). Such processes serve to dissipate excitation energy in excess of that required for photosynthetic metabolism (Long et al., 1994; Asada, 1999; Mullineaux and Karpinski, 2002; Bechtold et al., 2005). Failure to quench excess excitation energy leads to over-reduction of components of the electron transport chain (Foyer et al., 1997). Under such conditions, chlorophyll triplet states form in PSII and react with oxygen to form singlet oxygen (1O2), leading to PSII reaction centre damage and increased rates of lipid peroxidation (Hideg et al., 2002; Montillet et al., 2004; Krieger-Liszkay, 2005; Kruk et al., 2005; Flors et al., 2006).
The ROS, 1O2, O2·–, and H2O2, generated in the chloroplast during high light (HL) stress have been implicated as triggers of signalling pathways that influence expression of nuclear-encoded genes which may initiate acclimation processes or trigger cell death responses, depending on the degree of photo-oxidative stress suffered (Karpinski et al., 1997, 1999; Fryer et al., 2003; Op den Camp et al., 2003; Mateo et al., 2004; Danon et al., 2005). In previous studies the focus has been on the regulation of ASCORBATE PEROXIDASE2 (APX2, At3g09640), which encodes a cytosolic isoform of the enzyme (Santos et al., 1996) and whose expression is confined, in HL-stressed Arabidopsis, to bundle sheath tissue that surrounds the vasculature (Fryer et al., 2003; Ball et al., 2004; Mullineaux et al., 2006). When Arabidopsis is exposed to a 10-fold photoinhibitory excess light (EL) stress, APX2 expression can be observed in 15 min (Karpinski et al., 1997), has been associated with accumulation of H2O2 in bundle sheath cells (Fryer et al., 2003), and is dependent on active photosynthetic electron transport (Karpinski et al., 1997, 1999; Chang et al., 2004). APX2 expression is partially inducible in H2O2-treated leaves under low light (LL) conditions, such treatments conferring some protection against HL stress (Karpinski et al., 1999). From such data, it has been suggested that H2O2 sourced from bundle sheath tissue can act as both a local and a systemic signal to elicit defensive or acclimatory responses to environmental stresses (Karpinski et al., 1999; Fryer et al., 2003; Chang et al., 2004; Mateo et al., 2004; Bechtold et al., 2005; Mullineaux et al., 2006).
Under more moderate HL (2.5- to 5-fold above growth light intensity, hereafter called photosynthetically active photon flux density; PPFD), APX2 expression is also dependent on leaf water status (Fryer et al., 2003). This is associated with a rise in foliar abscisic acid (ABA) content in HL-stressed Arabidopsis leaves and induction of APX2 expression in ABA-treated leaves (Fryer et al., 2003; Rossel et al., 2006). Furthermore. a mutant with elevated expression of APX2, alx8-1 (altered expression of APX2 8-1), has double the foliar ABA content of wild-type plants, providing evidence of a direct link between ABA and the regulation of APX2 expression (Rossel et al., 2006). In addition, heat stress, which can be a component of HL stress in some experiments (Rossel et al., 2002), and heat shock transcription factor (HSF) gene expression may have a strong influence on the expression of APX2 and other members of this gene family (Panchuk et al., 2002; Davletova et al., 2005a).
Given the range of environmental factors influencing APX2 expression, the question can be posed: do other HL-responsive genes respond to all of the environmental and physiological cues that regulate APX2 expression? This is a particularly relevant question when it is considered that APX2 expression is specific to bundle sheath cells that clearly have a different ROS metabolism from that of neighbouring leaf tissues (Fryer et al., 2003; Mullineaux et al., 2006). To address this question, a strategy was adopted of selecting HL-responsive genes, irrespective of the functions they might code for, from a limited microarray experiment carried out on EL-stressed plants (O Richard and PM Mullineaux, unpublished data; see Supplemenatry Table S1 at JXB online) and then setting up a series of studies to determine how these genes would respond to the signals that are known to be important in APX2 expression.
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Materials and methods |
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Arabidopsis thaliana accessions, mutants, and transgenic lines
The mutants flu1-1, csd2-1, alx8-1, and atrbohD/F have been described previously (Torres et al., 2002; Op den Camp et al., 2003; Rizhsky et al., 2003; Rossel et al., 2006). The authors are grateful for kind gifts of flu1-1, csd2-1, and atrbohD/F seed from Professors Klaus Apel (ETH, Zurich), Ron Mittler (University of Nevada, Reno), and Jonathan Jones (Sainsbury Laboratory, Norwich), respectively. Where wild-type plants were used, their genotype was Col-0.
Plant growth and treatments
Wild-type plants and the mutants, except flu1-1, were grown in a controlled environment chamber in an 8 h photoperiod at 22 °C, 50% relative humidity (RH), and a PPFD of 150 µmol m–2 s–1. flu1-1 (Op den Camp et al., 2003) was grown under permissive conditions of 22 °C, 55% RH, and 100 µmol m–2 s–1 in a 24 h photoperiod. The induction of the 1O2 -producing phenotype was by transfer to a 12 h dark:12 h light cycle (non-permissive conditions) and carried out as described by Flors et al. (2006). A severe EL stress and a milder HL stress were imposed on plants by exposing them for 45 min to a PPFD of 2000 µmol m–2 s–1 and 750 µmol m–2 s–1, respectively. The type of lamp used in conjunction with a heat filter (water to a depth of 5 cm) has been described previously (Karpinski et al., 1999). Five- to six-week-old Arabidopsis thaliana Col-0 plants were used for all the treatments described. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) was diluted in 1% (v/v) Tween-20® to a final concentration of 10 µM and sprayed at the beginning of the 16 h dark period. A 45 min HL treatment was given to DCMU-treated and control plants 3 h after the onset of the light period. H2O2 (10 mM) and ABA (100 µM) were fed through the transpiration stream of detached fully expanded leaves as previously described (Karpinski et al., 1999; Fryer et al., 2003). The heat stress consisted of a 6 °C rise in growth temperature for 48 h at growth PPFD. RH was adjusted to maintain the same vapour pressure around the plants. Plants were confirmed as responding to the treatments by measurement of the dark-adapted chlorophyll a fluorescence parameter Fv/Fm using a fluorescence imaging instrument (Fluorimager, Technologica, Colchester UK; Barbagallo et al., 2003).
RNA isolation and real-time qRT-PCR
Immediately after treatments, leaves were harvested and frozen in liquid nitrogen. Total RNA was extracted from a pool of three plants using the RNeasy Plant Mini KitTM(Qiagen) according to the manufacturer's instructions. A minimum of two replicates of three pooled plants each from separate experiments were carried out for each treatment. For cDNA synthesis for real-time quantitative (q)RT-PCR, 3 µg of total RNA was treated with RNase-free DNase (Ambion) according to the manufacturer's instructions and reverse transcribed as previously described (Ball et al., 2004). Absence of genomic DNA contamination was confirmed by PCR using ACT1 (actin) primers (Ball et al., 2004). The efficiency of cDNA synthesis was assessed by real-time PCR amplification of control genes encoding actin (Ball et al., 2004). Real time qRT-PCR was performed using a cybergreen fluorescence-based assay as described previously (Ball et al., 2004). Gene-specific cDNA amounts were calculated from threshold cycle (Ct) values, expressed as relative to controls, and normalized with respect to actin cDNA according to Gruber et al. (2001). The primers used in qRT-PCR are given in Supplementary Table S2 at JXB online. All PCR products described in the text were checked by sequence analysis.
H2O2 determinations
H2O2 determination in acid extracts from leaves was done by the method described by Creissen et al. (1999).
Analysis of publicly available microarray data sets
Thirty-one publicly available microarray data sets from 22 experiments in which Arabidopsis was subjected to stress or treatments with signalling compounds, or in mutants defective in hormonal signalling, were analysed. These data sets are located at the Nottingham Arabidopsis Stock Centre (NASC) website (http://affymetrix.arabidopsis.info/narrays/experimentbrowse.pl). Experiments chosen were as follows; ein2-1 relative to wild-type [NASC experiments (exp.) #1, #2, #3, #10, and #52]; treatments with ACC (NASC exp. #172), ethylene fumigation (NASC exp. #37), ethylene inhibitors (NASC exp. #174), salicylic acid (NASC exp. #175), methyl jasmonate (NASC exp. #176), ABA (NASC exp. #57 and #188), a time-course series after indole acetic acid (IAA) treatment (NASC exp. #192), nitric oxide fumigation (NASC exp. #16); H2O2 treatment (NASC exp. #338), ozone fumigation (NASC exp. #26), UV-B (NASC exp. #163), heat (NASC exp. #146), cold (NASC exp. #138), salinity (NASC exp. #140), virulent Pseudomonas syringae pv. tomato DC3000 (NASC exp. #120), and far red, red, and blue light treatments (NASC exp. #124). Experimental statistics and normalization were carried out by NASC using MAS 5.0 scaling or by TAIR (The Arabidopsis Information Resource) using the print-tip-group lowess method. Normalized signal values were used to generate ratios of treated or mutant plants to untreated or wild-type plants in the same experiment. NASC-generated detection call and P-values were used to evaluate whether a gene was induced, suppressed, expressed but non-responsive, or not expressed (in both control and treatment). For co-regulation analysis, the total number for each class of regulation call (induced, suppressed) in each treatment was prepared for the entire array of 20 000+ genes and for the 27 HL-responsive genes (Table 1). Statistical comparison of these classes was done using a 
2 test comparing genes in the small group of 27 with the entire array as a whole. 2 P-values <0.05 were considered a weakly significant correlation, and those <0.001, a strongly significant correlation.
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HL-responsive genes
From a limited microarray experiment (see Supplementary Table S1 at JXB online) and subsequent confirmation by real-time qRT-PCR, the transcript levels of 28 genes, including APX2, were shown to be altered in leaves subjected to a severe EL stress of 2000 µmol m–2 s–1 for 45 min relative to LL controls (Table 1). The EL stress would have induced some heat stress and leaf drying (see Introduction) and it was reasoned that this set of genes might include those responsive primarily to these stresses. Therefore, a milder HL treatment of 750 µmol m–2 s–1 for 45 min was used (for a description of EL and HL treatments, see Materials and methods) and transcript levels relative to those of LL controls were determined by qRT-PCR for these genes. Expression of these genes, as well as APX2, was significantly altered by the HL treatment (Fig. 1A).
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0.5, respectively. In all cases, the highlighted values were significantly different from controls at least at the 95% confidence interval (P An important consideration in the interpretation of the data presented below is to consider if, like APX2, the expression of these 27 HL-responsive genes may be limited to bundle sheath cells. By analysing RNA from petioles and the upper third of a leaf, the relative transcript levels of 27/28 of the HL-responsive genes were measured under LL conditions (Table 1). In this analysis, the assumption was made that petioles contain a higher percentage of bundle sheath cells than distal leaf tissue. This analysis indicated that no gene is likely to be expressed exclusively in bundle sheath tissue, although there was clear increased induction in petioles in 25% of the genes examined (Table 1).
Publicly available microarray data sets from the NASC website were used to determine if the 28 HL-responsive genes (Table 1) were altered in their expression in response to a wide range of environmental stimuli (Table 2). The data showed highly significant induction of expression of these genes in response to ozone fumigation, H2O2 and ABA treatments, salinity, cold, heat, and light quality. Further, there was a lowering of gene expression in both nitric oxide-treated plants and in those infected with a virulent bacterial pathogen.
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Photosynthesis and the expression of EL-responsive genes
DCMU is an effective inhibitor of photosynthetic electron transport (Duysens, 1972) and of APX2 expression under HL conditions (Karpinski et al., 1997, 1999; Chang et al., 2004). Twenty-two of the 28 HL-responsive genes (81%; including APX2) were altered
2-fold in DCMU-treated plants subjected to HL relative to water-treated LL controls (Fig. 1B).
Chloroplastic sources of ROS and their effects on EL-responsive gene expression
The mutant flu1-1 accumulates 1O2 when transferred from growth in 24 h light (permissive conditions) to a 12 h dark period followed by re-exposure to the light (non-permissive conditions; Flors et al., 2006). The transcript levels of nine HL-responsive genes, excluding APX2, were induced in flu1-1 in response to a transfer from permissive to non-permissive conditions (Fig. 1C).
Under HL conditions, the H2O2 generated by CSD2, the plastidial Cu/Zn SOD isoform that catalyses dismutation of O2·– resulting from the photoreduction of O2 (Asada, 1999), may induce APX2 expression (Fryer et al., 2003). A HL treatment was carried out on csd2-1 that has 
60% less expression of CSD2 (At2g28190; Rizhsky et al., 2003). Under the growth conditions used (see Materials and methods), csd2-1 plants had paler leaves in the centre of the rosette compared with the wild type, but these leaves attained a normal appearance as they matured. This visible phenotype was reflected in smaller differences in chlorophyll content between mutant and wild-type plants as the leaves matured (see Supplementary Table S3 at JXB online). Accordingly, under HL conditions, gene expression was measured from outer leaves (leaves 1–3; see Supplementary Fig S1 at JXB online). Fifty-seven per cent (16/28) showed altered transcript levels in HL csd2-1 compared with HL wild-type plants (Col-0; Fig. 1D), with six genes (including APX2) having lower expression levels than HL wild-type leaves.
Responses to extraplastidic H2O2
Seven HL-responsive genes (25%), including APX2, responded to H2O2 fed to leaves through their transpiration stream (Fig. 2A). Of these seven genes, only At3g22840 (ELIP1), At1g53540 (HSP17.6-C), and APX2 were induced by both exogenous H2O2 and HL (Figs 1A, 2A). it was reasoned that this may reflect that few of the EL-responsive genes would respond to extracellular (apoplastic) or plasma membrane-produced H2O2. This notion was tested further using a double-null mutant lacking expression of the genes coding for the respiratory burst oxidases D and F (atrbohD/F), which are regarded as one of the main sources of ROS at the plasma membrane in leaf tissues (Torres et al., 2002). The expression of HL-responsive genes in atrbohD/F was unaffected, apart from APX2, At3g23990, and At1g35710 (Fig. 2B).
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A second approach used to increase extraplastidic H2O2 was the elevation of the growth temperature from 22 °C to 28 °C for 48 h. This treatment induced APX2 expression (Fig. 2C) and resulted in a 3.4-fold increase in foliar H2O2 levels (49.8±16 nmol g–1 FW compared with 171.7±19.5 nmol g–1 FW, respectively; n=6, ±SE). Apart from APX2, no other HL-responsive genes were altered in a pattern consistent with their response to the HL treatment (Fig. 2C). This mild heat treatment was not associated with any visible damage to rosettes either during or after the heat stress (data not shown).
Responses to ABA
The expression of the majority (68%) of the HL-responsive genes was responsive to treatment of plants with ABA (Fig. 3A). Thirty-nine per cent of the HL-responsive genes also showed 2-fold raised transcript expression in the mutant alx8-1 (Fig. 3B).
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Discussion |
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In the natural environment, sudden exposure of leaves to full sunlight may result not only in photoinhibition but also in stress due to an increase in temperature and a decline in leaf water potential (Schultz and Matthews, 1997; Singaas et al., 1999; Valladares and Pearcy, 2002; Proctor, 2003). This is in contrast to experimentally imposed HL stress. In such studies, it is usual to attempt to minimize the effects of other factors to clarify interpretation of data (Karpinski et al., 1997, 1999; Rossel et al., 2002; Davletova et al., 2005a, b). This has implications for understanding how a sudden increase in light intensity in the natural environment may be perceived and responded to. This question is particularly pertinent to Arabidopsis, a plant that can adapt well to a fluctuating light environment and is tolerant of a range of light intensities (Kulheim et al., 2002; Muller-Moulé et al., 2004; Bechtold et al., 2005).
Signal transduction involving photosynthesis and chloroplastic sources of ROS
Like APX2, most of the 27 genes showed a dependency on photosynthesis for their response to HL, as evidenced by an opposite response in leaves treated with the photosynthesis inhibitor DCMU (compare Fig. 1A with B). Inhibition of photosynthetic electron transport by DCMU would block the Mehler reaction as a source of O2·– and H2O2, and cause increased production of 1O2 (Duysens, 1972; Basra et al., 2002; Fufezan et al., 2002; Fryer et al., 2003; Krieger-Lizskay, 2005; Flors et al., 2006). Further, HL and DCMU treatment, alone or in combination, promotes the production of 1O2 in PSII reaction centres. The reactivity of this ROS excludes its diffusion from the site of production (see Introduction, Fufezan et al., 2002; Krieger-Liszkay, 2005; Flors et al., 2006). Therefore, this suggests that 1O2 produced in plants under HL could not be involved in controlling the expression of the HL-responsive genes. However, the observations on the expression of these genes in flu1-1 under 1O2-producing conditions (Fig. 1D) suggest that a more complicated situation exists. The site of 1O2 production in the flu1-1 mutant is the chloroplast stroma because the accumulated photodynamic compound, protochlorophyllide (Op den Camp et al., 2003), will not have been assembled into photosystems. Thus 1O2 produced in response to HL/DCMU and the flu1-1 mutant may have a different origin within the chloroplast. Taken together, these data suggest that the origin of the 1O2 may determine the types of genes it can induce and that signalling initiated by 1O2 in flu1-1 may not reflect a similar signalling role for this ROS in wild-type plants.
A major source of chloroplastic H2O2 has been proposed to be the Mehler reaction coupled to the dismutation of O2·– and catalysed by a thylakoid-associated Cu/Zn SOD isoform (CSD2; see Introduction; Asada, 1999; Ort and Baker, 2002). Under stress conditions, this source of H2O2 has been suggested to be a source of signalling for altering gene expression in response to environmental stress (Mullineaux and Karpinski, 2002; Fryer et al., 2003; Chang et al., 2004). The lowered transcript levels of six genes, including APX2, in HL-treated csd2-1 (Rizhsky et al., 2003) compared with wild-type plants (Fig. 1C) does support the notion that H2O2 sourced from the chloroplast may be part of a signalling system regulating the expression of at least some HL-responsive genes (Karpinski et al., 1997, 1999; Fryer et al., 2003; Mullineaux et al., 2006). However, a further 10 of the EL-responsive genes showed greatly increased transcript levels in the HL-stressed mutant compared with the wild type (Fig. 1C), perhaps suggesting that these genes are responsive to more subtle changes in chloroplast ROS metabolism in the mutant (Rizhsky et al., 2003). For example, partial loss of the CSD2 isoform in csd2-1 may be compensated for by increased expression of other members of the gene family, such as FSD1 (At4g25100) coding for stromal Fe-SOD (Rizhsky et al., 2003).
Photorespiration has been ruled out as an indirect source of H2O2 for signalling in the regulation of APX2 expression (Fryer et al., 2003). For the other HL-responsive genes, only At1g53540, At5g52640, and At2g29500 (Table 1) show altered expression in microarray-based studies on catalase-deficient Arabidopsis at growth PPFD (Vanderauwera et al., 2005), which could indicate responsiveness to photorespiratory H2O2. However, these three genes were induced in non-permissive flu1-1 (Fig. 1D); one of these (At2g29500) showed suppressed induction in HL-treated csd2-1 (Fig. 1C), while At1g53540 was responsive to exogenously supplied H2O2 (Fig. 2A). These data suggest that these three genes are responsive to ROS from numerous sources, a situation reported for many more oxidative stress-responsive genes in a recent meta-analysis of ROS-related microarray data (Gadjev et al., 2006). In support of this proposition, immersion of young seedlings in 20 mM H2O2 induced a significant response from this group of 27 HL-responsive genes when assayed using microarrays (Table 2). Similarly, other treatments (cold, salinity, ozone fumigation), that might be expected to elicit oxidative stress and production of a range of ROS (Mullineaux and Karpinski, 2002; Joo et al., 2005; Davletova et al., 2005b), did cause this cohort of genes to respond significantly (Table 2).
The data presented here confirm and extend the importance of a direct and characteristic requirement for active photosynthetic electron transport in the regulation of HL-responsive genes (Fig. 1B; Escoubas et al., 1995; Karpinski et al., 1997, 1999; Rossel et al., 2002; Steinbrenner and Linden, 2003), but this did not necessarily equate with a requirement for ROS as a signalling molecule (Fig. 1C, D). This suggests that additional signals exist that connect changes in the activity of photosynthetic electron transport to altered expression of nuclear genes. Signalling could be initiated by changes in the redox state of the plastoquinone pool (Escoubas et al., 1995; Karpinski et al., 1997, 1999; Rossel et al., 2002; Fryer et al., 2003; Steinbrenner and Linden, 2003), although this has been questioned for induction of APX2 (Fryer et al., 2003). A further possibility could include altered levels of signalling molecules whose biosynthesis occurs wholly or partly in the chloroplast, such as salicylic acid, ABA, and reduced glutathione (Bechtold et al., 2005; Mateo et al., 2006; Mullineaux et al., 2006).
Extracellular H2O2 is a signal for HL-mediated induction of APX2
The feeding of 10 mM H2O2 through the transpiration stream induces APX2 expression, can protect leaves against subsequent photoinhibition, and does not per se promote foliar oxidative damage (Karpinski et al., 1999). Therefore, it was surprising, especially in the light of the survey of microarray experiments of H2O2-treated seedlings (Table 2), that only APX2 and two other genes were affected in a manner consistent with their altered expression under HL conditions (Figs 1A, 2A). Thus under H2O2 treatments that do not promote oxidative stress, this ROS may be an important factor in transducing signals for inducing APX2 expression, but not for a large majority of the HL-responsive genes examined here.
From H2O2 feeding experiments done here and previously (Karpinski et al., 1999), it was reasoned that APX2 expression might be particularly responsive to extracellular sources of ROS at the plasma membrane and in the apoplast. Recently, plasma membrane-localized NADPH oxidases, which catalyse the production of ROS, have been shown to be important in the induction of stress-responsive genes in Arabidopsis suffering ozone fumigation or EL (Davletova et al., 2005a; Joo et al., 2005). Under the HL conditions used in this study, the diminished expression of APX2 and one other gene (At1g35710) in the AtrbohD/F mutant (Fig. 2B) suggests that NADPH oxidases may be part of a signalling route controlling APX2 expression, similar to that described for APX1 (Davletova et al., 2005a). Increased expression of APX2, but none of the other HL-responsive genes, in mild (28 °C) heat-treated wild-type plants (Fig. 2D) suggested that H2O2 from extracellular sources is an important component in the regulation of APX2 expression. The mild heat treatment also raised foliar H2O2 levels, consistent with previous reports (see Results; Dat et al., 1998; Vallelian-Bindschedler et al., 1998; Rivero et al., 2004; Larkindale and Huang, 2005) and, while its source in this case has not been investigated, other studies have suggested that the plasma membrane or the apoplast is the origin of this increase during heat stress (Larkindale and Huang, 2005; Volkov et al., 2006).
It is important to note that in both experiments, the expression of none of the seven heat shock genes included in this study (Table 1) was induced (Fig. 2C). While this is in contrast to the response of these genes to a severe heat shock (38 °C; Table 2), it is consistent with previous reports showing that plant heat shock genes do not respond to moderate changes in temperature (Wu et al., 1988; Conner et al., 1990). Further, the induction of heat shock genes by HL was shown to be due to the light intensity and not the associated increase in temperature (Rossel et al., 2002). In summary, APX2 was the only gene examined here whose expression was consistently affected by treatments that affected apoplastic H2O2 or in plants with altered capacity to produce plasma membrane/apoplast-sourced H2O2 (Fig. 2A–C). These data imply that any signalling route controlling APX2 expression may include the activation of H2O2 production at the plasma membrane of bundle sheath cells and suggest that H2O2-mediated control of APX2 expression in bundle sheath cells is fundamentally different from that in other leaf tissues.
ABA in HL stress-responsive gene expression and its relationship with H2O2
Sixty-one per cent of the HL-responsive genes were altered in their expression in ABA-treated leaves, as described previously for APX2 (Fig. 3A; Fryer et al., 2003), although in alx8-1, with raised steady-state levels of ABA and constitutive expression of APX2 (Rossel et al., 2006), the number of genes altered in their expression was diminished (Fig. 3B). These data confirm those in publicly available microarray data sets (Table 2). ABA has recently been shown to accumulate in wild-type leaves in response to HL (Rossel et al., 2006), which together with the data presented here suggests that ABA plays a central role in signalling HL-responsive gene expression, emphasizing an increased linkage between leaf water potential and the capacity to dissipate excitation energy (Fryer et al., 2003).
ABA is thought to act as a signal in the acclimation to a range of abiotic stresses (Chandler and Robertson, 1994; Siddiqui et al., 1998; Hare et al., 1999) and coincides with the findings of increased H2O2 levels after ABA treatment (Jiang and Zhang, 2001; Guan et al., 2000; Pei et al., 2000) as well as the induction of antioxidant gene expression such as those coding for SODs (Sakamoto et al., 1995; Kaminaka et al., 1999) and catalases (Guan et al., 2000). The picture emerging from this and previous studies suggests that ABA- and H2O2-mediated signalling can be integrated for many different leaf cell types and not just for guard cells (Pei et al., 2000; Murata et al., 2001; Mullineaux et al., 2006).
Limitations in the interpretation of whole leaf gene expression data
In contrast to APX2, for the other 27 HL-responsive genes in this study, information on tissue-specific expression profiles using in situ detection of mRNAs or from promoter–reporter gene fusions in Arabidopsis is sparse. However, by extracting RNA from the petioles and the upper third of HL-exposed leaves, some indication of enrichment of expression in the vasculature could be gained if, in petioles, steady-state RNA levels in response to HL were greater than those in the distal leaf blades (Table 1). It was reasoned that the upper third of the leaf blade would contain a lower number of bundle sheath cells than petioles. Interestingly, of the genes examined, induction in petioles compared with leaf blade was very different (ratios >10) for six genes, suggesting that there is preferential expression for 25% of the HL-responsive genes, including APX2 in petioles (Table 1). Of these, two genes (At2g29500 and At1g53540) coding for small heat shock proteins show a particularly strong preference for expression in petioles, greater than the values for APX2 (Table 1). These data indicated that the petioles responded much more strongly to HL compared with leaf blade tissue, lending support to the proposal that bundle sheath cells are more responsive to fluctuating light levels than mesophyll cells (Fryer et al., 2003).
While the differences in response to the treatments described above between APX2 and the remaining 27 HL-responsive genes may reflect an operation of different HL-activated signalling routes, it is only really possible to conclude that regulation of APX2 is different from the overall response of the other HL-responsive genes examined in the absence of detailed information on tissue specificity of expression. Nevertheless, it is at least noteworthy from this study that the heat shock protein gene, At1g53540, with the greatest expression values in petioles, most closely matches APX2 in its response to mutants and treatments (Figs 1–3
). However, in general from this study, if genes are expressed in more than one tissue of the leaf, then the measured value of their expression from total leaf RNA will be an aggregate of different levels of expression in different tissues. Therefore, the operation of distinct tissue-specific signalling pathways cannot necessarily be unambiguously observed in most cases from these kinds of studies. This is the case for at least 75% of the HL-responsive genes, with the only clear exception of APX2, and is an issue for more extensive microarray-based studies than the limited case study described here (see, for example, Gadjev et al, 2006). This lack of information on the tissue specificity of stress-responsive genes seriously hampers the construction of any useful and robust models that rely upon interpretation of transcriptomics data, and is in urgent need of resolution.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Table S1. DE values, SAM scores of excess light-responsive genes from a limited microarray study. This is preceded by a description of the microarrays and data analysis.
Table S2. Oligonucleotide primer sequences used in this study.
Table S3. Chlorophyll a and b contents in the inner and outer leaves of Col-0 and csd2-1 rosettes.
Fig. S1. Chlorophyll fluorescence imaging of the quantum efficiency of PSII (Fq'/Fm') showing the difference between outer leaves (numbered 1–3) and inner leaves (4–7).
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Acknowledgements |
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The authors are grateful to Professors R Mittler, K Apel, and J Jones for gifts of mutants. PMM gratefully acknowledges the support of the University of Essex and the Biotechnology and Biological Sciences Research Council (grant number 208/P18073). CG acknowledges the support of a John Innes Foundation research studentship.
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* Present address: Dipartimento di Colture Arboree, Università di Bologna, Viale G. Fanin 46, 40127 Bologna, Italy.
Present address: Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario. London, Ontario, Canada N6A 5C1.
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