Generation of O
, photosynthetic activity and antioxidative response of Arabidopsis plants
Two-week-old Arabidopsis seedlings grown on soil were treated with MV in the light followed by NBT infiltration to visualize O
formation. Superoxide anion is the major oxidant species responsible for reducing NBT to insoluble formazan (Maly et al. 1989), which is observed as a purple deposit. Numerous localized spots were observed in the leaf after a 2 h MV treatment, and they spread all over the leaf by longer MV incubations (Fig. 1). A representative Arabidopsis seedling at 2 h MV treatment after NBT staining is also shown (Fig. 1). By contrast, plants subjected to the same MV treatment for 2 h did not accumulate H2O2 at a detectable level as revealed by DAB staining (Fig. 1).
To test the functional performance of the photosynthetic apparatus after MV treatment, we measured the chlorophyll fluorescence emitted by Arabidopsis leaves after saturating pulses of halogen actinic light. These pulses fully reduce photosynthetic electron carriers and several parameters can be measured, such as Fv/Fm, indicating the reduction state of the primary electron acceptor of PSII (QA); qP, reflecting the PSII operating efficiency; and NPQ, which represents the energy dissipated as heat related to energization of the thylakoid membrane due to lumen acidification during photosynthesis. Table 1 shows that the photosynthetic performance of leaves of soil-grown Arabidopsis plants was better than that of plants cultured on solid MS medium plus sucrose. When plants grown on soil were subjected to MV treatment Fv/Fm decreased steadily with time after the MV treatment, indicating a change of the electron transfer capacity. Concomitantly with this, the ΦPSII of electron transfer at PSII, which indicates the photosynthetic efficiency of PSII, was markedly reduced at 3 h MV treatment. The PSII redox state (1−qP) was higher, to various degrees, in all MV-treated plants compared to non-treated plants, and markedly increased at 3 h MV treatment (Table 1), suggesting that the reduced state of QA diminished under prolonged MV treatment. Within the first hour of MV exposure, the NPQ of these leaves was 22% higher than in control leaves, tending to decrease after several hours of MV treatment, possibly due to decreased electron transfer capacity.
Electrolyte leakage, indicative of membrane integrity, and photosynthetic pigments were measured at different time points after MV exposure and the results are shown in Supplementary Fig. 1. From the beginning of the MV treatment, ion leakage was detectable to different extents in comparison to control Arabidopsis leaves, increasing markedly at the end of the treatment (70 % at 7 h) (Supplementary Fig. 1A). Total chlorophyll and carotenoid contents were not affected within 4 h of MV challenge (Supplementary Fig. 1B and C).
The antioxidant response of Arabidopsis plants under MV treatment was followed by determining SOD, CAT and POD activities in native protein gels. Two-week-old Arabidopsis plants were exposed to 50 μM MV for up to 6 h, and leaf proteins extracted at different time points. SOD activity was relatively constant during the oxidative challenge (Supplementary Fig. 2). After 6 h MV treatment CAT activity was invariable, while POD activity decreased in leaf extracts (Supplementary Fig. 2). APX, GR and CAT activities were assayed in solution (Supplementary Fig. 3) and no variations were observed between control and 2 h MV treated plants. After longer MV exposure times there was a significant drop in APX activity, remaining a total activity of 74 % at 4 and 6 h comparing with that of control plants. On the other hand, GR and CAT were unaffected in response to longer MV exposure. These results suggest that the antioxidant machinery of Arabidopsis leaves was largely unaltered by 2 h MV treatment, although at longer exposure times, peroxidases were the most affected enzymes.
Consequences of MV treatment of illuminated Arabidopsis seedlings on protein levels
Chloroplast early ROS targets such as GS and Rubisco (Palatnik et al. 1999) were followed in MV-treated plants by GS in-gel activity measurements and Western blot analysis (Fig. 2). GS protein and activity remained constant during 6 h of MV treatment, but after 24 h of oxidative challenge no GS activity was detected with no significant modification of GS protein level (Fig. 2a, b). Rubisco large subunit and presumably a degradation product visible since 1 h MV exposure remained constant over 24 h of oxidative treatment (Fig. 2c).
Two nuclear-encoded chloroplast proteins such as Hsp70 and 2-Cys Prx and a cytosolic Hsp70 were also analyzed by immunoblotting based on the observations that some members of the Hsp70 family are stress-inducible (Boston et al. 1996), and that 2-Cys Prx prevents oxidative damage of chloroplast proteins (Konig et al. 2003). Chloroplastic and cytosolic Hsp70 were specifically detected using different antibodies that discriminate between them (Rial et al. 2003). Cytosolic Hsp70 remained unaltered for up to 6 h of MV treatment in the light (Fig. 2d), while chloroplastic Hsp70 was clearly induced at 2–4 h of MV treatment (Fig. 2e). After 24 h of MV treatment, Hsp70 declined in both compartments (Fig. 2d, e). On the other hand, the levels of 2-Cys Prx were constant for the first 2 h of oxidative treatment, after which the enzyme decreased markedly becoming undetectable at 24 h (Fig. 2f). The 2-Cys Prx monomer was visible at 1–2 h of oxidative stress, probably arising from overoxidation of 2-Cys-Prx monomer as it was reported in barley plants subjected to drought or low temperatures (Konig et al. 2003).
These results show that a 2 h MV treatment of Arabidopsis leaves in the light generates only mild changes in the levels of proteins under analysis, suggesting that main cell functions are largely preserved under this experimental condition.
Transcriptional response of Arabidopsis seedlings to MV treatment in the light
As shown, several hours of steady generation of O
in illuminated chloroplasts of Arabidopsis seedlings caused severe oxidative damage. We wanted to analyze transcriptional changes occurring at earlier time points in response to chloroplastic O
generation, i.e., when inhibition of photosynthesis is incipient (Table 1) and the level and activity of chloroplastic (Fig. 2) and antioxidant proteins (Supplementary Figs. 2 and 3) are not affected. To this end, two-week-old Arabidopsis seedlings were removed from soil and floated for 2 h in the light in a solution containing 50 μM MV (in the presence of 0.01% (v/v) Tween 20 to facilitate cellular uptake of MV). Control plants were incubated in the same way with the exception that MV was omitted. RNAs were isolated from the aerial parts of MV-treated and control seedlings, and subjected to transcript profiling employing Affymetrix GeneChip array ATH1 (for more details of array experimental design, data normalization and analysis see “Materials and methods”). The microarray data have been deposited in the public repository ArrayExpress (www.ebi.ac.uk/arrayexpress) under the accession number E-ATMX-28. From all genes analyzed, the vast majority exhibited increased abundance taking a minimal 3-fold difference between MV-treated and control samples as a threshold (Supplementary Table 1).
Microarray data were verified by Northern blot analysis (Fig. 3) of a selection of six genes strongly induced in the profile such as transcription factors and Hsp genes. In all cases, a clear increase in transcript abundance was observed at 2 to 4 h of MV treatment, which is in agreement with the expression data obtained through the gene chip hybridization experiments. Except for Hsps, all transcripts assayed were down-regulated after 24 h of MV treatment.
Functional analysis of genes early induced by MV treatments
The potential functions of early induced genes by MV in different biological processes were assessed by the MapMan software. A large fraction of genes exhibiting an at least 3-fold change in expression level were found to be up-regulated after the MV treatment (Table 2). Sixty-eight of the MV-induced genes have no ontology assigned or unknown function (Table 2). Several of the MV-affected genes belong to categories that function in abiotic or biotic stress responses, that encode enzymes that have a role in transcriptional regulation or are involved in signalling events (Table 2, Supplementary Fig. 4 and 5). Among the genes belonging to abiotic stress category, those encoding Hsp were the most abundant (Table 3 and Supplementary Fig. 4). From the 18 highly induced Hsp genes, ten belong to the small Hsp family, five members co-localize in the cytosol, and nine genes still have undetermined subcellular location (Table 3). Protein kinases, protein phosphatases, and receptor kinases (mainly leucine rich repeat) were highly induced among the genes involved in signalling processes (Supplementary Fig. 5). Importantly, the expression level of various transcription factors, in particular those of the WRKY family, increased after a short-term (2 h) MV treatment (Table 4).
Only a small number of genes encoding proteins of redox-related metabolism, including glutaredoxin family proteins, and mono and dehydroascorbate reductases, were found to be affected by MV under our experimental conditions (Supplementary Fig. 4). These data are consistent with a preserved antioxidant response in Arabidopsis leaves at 2 h MV treatment, as suggested by the enzyme activity measurements (Supplementary Fig. 2 and 3).
Of the few genes exhibiting a minimum of 3-fold reduced expression upon MV treatment (Supplementary Table 1), five encode proteins of unknown function, four encode auxin-induced proteins (Supplementary Fig. 5A), two encode transcription factors (Table 4 and Supplementary Table 3), and two encode PSI and PSII proteins. These data are in accordance with the observed decrease in the photosynthetic electron transport activity of Arabidopsis leaves (Table 1). On the other hand, transcript of 9-kD protein of PSI showed an opposite pattern in a 24 h MV transcriptomic footprint (Gadjev et al. 2006), probably reflecting an adaptation of plants to long term MV treatment. Other genes involved in photosynthesis-related processes such as the Calvin cycle or photorespiration were not affected by the 2 h MV treatment (data not shown).
Down-regulation of genes related to the photosynthetic electron transport system were also observed when Arabidopsis plants were exposed to high light intensity (Kimura et al. 2003). This condition leads to the production of ROS in the chloroplast (Niyogi 1999). To investigate the transcriptional response of Arabidopsis under situations where ROS are produced in the chloroplast, we performed comparative analysis of the transcriptome of Arabidopsis seedlings subjected to 2 h MV treatment and high light, using publicly available microarray experiments (Rossel et al. 2002; Kimura et al. 2003; Vanderauwera et al. 2005). At a threshold of 2.8-fold change, Hsps were the most abundant up-regulated genes in common (Supplementary Table 2). Other common genes up-regulated codify for the cytosolic APX1 (At1g07890) and the transcription factors DREB2A (At5g05410) and the NAC domain protein (At1g01720). DREB2A is a key regulator of drought response, therefore 61 genes responsive to high light and drought (Kimura et al. 2003) were additionally compared with MV-regulated genes. Only three genes were in common among high light, drought and MV treatment of Arabidopsis seedlings encoding: an arabinogalactan-protein AGP2 (At2g22470), an Hsp70 (At3g12580) and a putative protein phosphatase 2C (At3g62260).
We further analyzed the expression of all 2 h MV induced Hsp genes (Table 3) under other abiotic stress conditions such as salt, drought, wounding or cold using GENESVESTIGATOR tools (Supplementary Fig. 6). Two Hsp70 (At1g16030 and At2g32120) and two small Hsps (At1g54050 and At5g51440) were not significantly induced by any stress conditions other than late oxidative or heat stress. On the other hand, the HsfA4A (At4g18880) was only up-regulated more than 3-fold by osmotic stress (Supplementary Fig. 6).
Transcription factors induced or repressed by 2 h MV treatment in the light
Several members of common transcription factor families such as AP2-ERF, C2H2, Hsf, NAC and WRKY were highly induced by MV (Table 4). MV-dependent up-regulation of members of the AP2-ERF (At1g22810 and At2g33710) and WRKY (At5g24110) families was confirmed by Northern blot analysis (Fig. 3). The highest number of MV-affected transcription factor genes was found in the WRKY family, with nine members being up-regulated at least 3-fold. Among the WRKY family members, WRKY6, WRKY30 and WRKY46 were highly stimulated (Table 4). Comparing the profiles of these transcription factors (Table 4) with transcription factor genes up-regulated by H2O2 exposure (Davletova et al. 2005) or generated in CAT-deficient Arabidopsis mutants exposed to high light conditions (Vanderauwera et al. 2005), rendered an overlap of eight genes in all three conditions (Supplementary Table 3). Several members belonging to the WRKY family were expressed in two out of three experiments and only WRKY6 was induced by the three oxidative treatments. WRKY46, WRKY28 and WRKY22 were up-regulated exclusively by MV treatment.
As mentioned above, only a relatively small number of genes was found to be down-regulated by MV treatment (Table 2). They include two transcription factors belonging to basic helix-loop-helix (bHLH) (At4g36540) and HB families, an homeotic protein BEL1 homolog (At5g41410) (Table 4).
Identification of conserved motifs in promoters of MV induced genes
Promoters of genes strongly up-regulated after MV treatment were selected for a MEME search to identify potential transcription factor binding sites. This computational tool discovers motifs (highly conserved regions) in groups of DNA or protein sequences, i.e., in promoters of genes selected according to a common expression pattern (Bailey and Elkan 1994).
For this analysis we selected all genes that exhibited at least 4-fold induction after MV treatment (168 genes in total). One-kb long promoter regions located 5´ upstream of the predicted ATG initiation codons of these genes were analyzed using the MEME software tool. Sixteen motifs were found and represented with different incidences in the dataset and in the genome (Supplementary Table 4). We could not identify with confidence known transcription factor binding motifs in the promoters of MV-inducible genes (Supplementary Table 4), except for the core ABRE element (ACGT) (Leung and Giraudat 1998), an abscisic acid-dependent cis-acting regulatory element, found at high frequency in motif 2 and less frequently also in motif 9 (Fig. 4). Instead, different motifs were found to be highly over-represented in genes encoding transcription factors, Hsps or Hsfs, and proteins involved in signalling cascades (Supplementary Table 4).
Motif 2 (CGGTCCACGTG) was found in eight genes encoding a putative Hsp70, a small Hsp17.4-CIII, WRKY6, a cytochrome P450 enzyme, a methyl transferase, an ethylene-responsive binding protein, and a Zn finger protein. Two of these genes contain additional motifs, i.e., the Zn finger protein gene (At5g27420) has motifs 1 and 7, and the methyl transferase gene (At1g21110) has motifs 7 and 16. Interestingly enough, this methyl transferase promoter shares motifs 2, 7 and 16 with the promoters of WRKY6, WRKY30 and WRKY46, respectively. Motif 9 (ACGACGTCGTTT) was found in the promoters of 23 genes encoding Hsp81, HsfA4A and several signal transduction components such as protein phosphatase 2C (also containing motifs 8 and 11), MAPK, and a leucine-rich repeat transmembrane protein kinase (Supplementary Table 4).
Motif 1 (AATGGGCCTTAA) or motif 5 (GAAAGTTCCAGA) was highly represented in promoters of Hsp genes (Supplementary Table 4), and both motifs were detected in small Hsps genes (At3g46230 and At1g54050). Motif 5 contained the consensus sequence ‘nGAAn’, a highly conserved heat shock element found in the promoters of many genes involved in cell protection such as protein folding, detoxification, and maintenance of cell wall integrity in Saccharomyces cerevisiae (Yamamoto et al. 2005).
Motifs 7, 8 and 11 were found in several transcription factors belonging to different families (WRKY, AP2 and NAM) (Supplementary Table 4). Remarkably, the four WRKY transcription factors highly induced by MV did not share common motifs in this analysis. The four different motifs that could be distinguished in the promoters of WRKY6 (motif 2, CGGTCCACGTC), WRKY11 (motif 14, CCGGTCACCTCC), WRKY30 (motif 7, GAAAAGTCAAAC) and WRKY46 (motif 16, CGTCGACCAG) showed p-values lower than 10−6 (Supplementary Table 4), indicating a low probability that a single random subsequence matches along the length of the motif. WRKY factors bind to W boxes (C/T)TGAC(T/C), which are present in the promoters of many plant genes that are associated with defense (Eulgem et al. 2000). Motif 7 (GAAAAGTCAAAC), which at high frequency contains the GTCA core element (Fig. 4) that resembled the inverted consensus W box sequence, was found to be present in the WRKY30 promoter (Supplementary Table 4). Motif 11 (AACGGTCAAGAT), shared by the promoters of three different transcription factors, also contains the GTCA element at high frequency (Fig. 4).
Leaf proteins bind to the GAAAAGTCAAAC motif
The presence of WRKY proteins in MV-treated Arabidopsis leaf extracts was assayed by gel retardation experiments. To this end, a 30-mer double-stranded oligonucleotide (W box) containing the consensus motif 7 was used as a probe (Fig. 5). Several retarded bands were observed when leaf protein extracts were tested for W box binding (Fig. 5a). Competition experiments using unlabelled W box and mutated W box (mW box) probes revealed that two of the retarded bands were due to specific binding of WRKY proteins present in leaf extracts (Fig. 5a). Specific binding activity to motif 7 increased after a 2 h MV treatment (Fig. 5b), indicating that WRKY proteins were rapidly induced by the oxidative treatment.
Rapid response of WRKY30 promoter containing the GAAAAGTCAAAC motif to oxidative treatments
To assess in vivo functionality of motif 7, we produced transgenic Arabidopsis plants expressing the GUS reporter gene under the control of 1.96-kb WRKY30 promoter (PrAtWRKY30::GUS lines), which contains this motif. WRKY30 expression was clearly induced by MV in the light (Fig. 3), as several other WRKY family members (Table 4). Therefore, we analyzed the response of WRKY30 to different oxidative treatments. PrAtWRKY30::GUS lines were subjected in the light to MV and to externally added hydrogen peroxide and the GUS staining pattern was recorded (Fig. 6). GUS gene under the control of WRKY30 promoter was highly and rapidly induced by MV, while 24 h of hydrogen peroxide treatment was necessary to detect GUS activity (Fig. 6).
Identification of MV-specific responsive genes in Arabidopsis leaves
In illuminated leaves, MV propagates superoxide anion in the chloroplast (Mehler 1951; Asada and Takahashi 1987), which subsequently dismutates to H2O2 either spontaneously or enzymatically via SODs (Tsang et al. 1991). To present more insight into the MV-specifically regulated genes in Arabidopsis leaves, we compared the 2 h MV expression profile with the one obtained after 1 h H2O2 treatment (20 mM), which was exogenously added to 5-day-old wild-type Arabidopsis (Davletova et al. 2005) or H2O2 internally generated in CAT-deficient plants subjected for 3 h to high light (Vanderauwera et al. 2005), following the threshold of 3-fold change. Figure 7 presents the overlap of the rapidly MV-regulated genes with H2O2-regulated genes. Eighty four genes were responsive to MV and H2O2 internally or externally generated. Within these 84 commonly regulated genes, several transcription factors could be identified (Supplementary Table 3) such as WRKY6, ZAT12, and DREB2A, being the last one also in common with high light stress (Supplementary Table 2). After identification of all genes in common among these stress conditions, we extracted the genes that were exclusively regulated by MV. We identified 136 genes, within them 117 genes were up-regulated, and nineteen genes encoded unknown proteins (Supplementary Table 5). We further compared the responsiveness of these up- and down-MV-regulated genes to various environmental stimuli by hierarchical average linkage clustering separately (Fig. 8 and Supplementary Fig. 7). Several groups of co-expressed genes were observed at diverse early and late stress conditions. Three clusters of early transcriptional changes (Fig. 8 I, II and III) could be distinguished for up-regulated genes in wounding, drought and osmotic stress, partially overlapping with late gene clusters responsive to wounding, oxidative and osmotic stresses. These clusters are dominated by genes encoding proteins of unknown functions. The expression profile of 2 h MV responsive genes was not affected by early salt, oxidative and genotoxic stresses. A remarkable group of genes is the one strongly regulated by 2 h MV but not responsive to any other environmental stress (Fig. 8, IV and Supplementary Fig. 7). This group is represented by fifteen genes up-regulated and seven genes down-regulated by MV. From the up-regulated genes not responsive to any other stress, there are six genes encoding protein of unknown functions, a quinone oxidoreductase-like protein, a putative S-adenosyl-l-methionine:trans-caffeoyl-Coenzyme A 3-O-methyltransferase, a wall-associated kinase 2, a disease resistance protein-like, three genes related with signal transduction pathways, an ethylene response factor-like AP2 domain transcription factor, and a putative SCARECROW gene regulator (Fig. 8). From the genes down-regulated by MV but not responsive to any other stress there are three which encode proteins of unknown functions, the PSI 9kDa protein and an homeotic transcription factor BEL1 (Supplementary Fig. 7).