Arabidopsis thaliana (L.) Heynh. ecotype Columbia-0 (Col-0, wild-type) constitutively expressing glycolate oxidase (GO, At3g14420) in the plastids (GO plants) under the cauliflower mosaic virus 35S promoter were generated in our previous work (Fahnenstich et al., 2008). In these plants to direct the expression of GO to the chloroplats the stromal targeting presequence from Arabidopsis thaliana phosphoglucomutase (At5g51820) was used (Fahnenstich et al., 2008). WT and GO transgenic plants were grown in pots containing 3 parts of soil (Gebr. Patzer KG, Sinntal-Jossa, Germany) and one part of vermiculite (Basalt Feuerfest, Linz, Austria) under a 16 h-light/8 h-dark regime at photosynthetically active photon flux densities (PPFD) of 75 μmol quanta m⁻² s⁻¹ at 22°C day/18°C night temperatures and a CO₂ concentration of 3000 ppm. After 3 weeks of growth plants were transferred to ambient CO₂ concentration (380 ppm) and the same PPFD. Whole rosettes were harvested at different time points after transfer, immediately frozen in liquid nitrogen and stored at −80°C until use for RNA isolation and H₂O₂ measurements.

For the large-scale qRT-PCR analysis, total RNA was extracted from 100 mg leaves (fresh weight) using RNeasy Plant Mini kit (Qiagen, Valencia, USA) according to the manufacturer's protocol. DNAse I digestion was performed on 20–30 μg of total RNA using TURBO DNase Kit (Ambion, Cambridgeshire, UK) according to manufacturer's instructions. RNA integrity was checked on 1% (w/v) agarose gels and concentration measured with a Nanodrop ND-1000 spectrophotometer before and after DNAse treatment. Absence of genomic DNA was confirmed subsequently by quantitative PCR using primers that amplify an intron sequence of the gene At5g65080 (forward 5′-TTTTTTGCCCCCTTCGAATC-3′ and reverse 5′-ATCTTCCGCCACCACATTGTAC-3′). First-strand cDNA was synthesized from 8 μg to 10 μg of total RNA using RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) following the manufacturer's protocol. The efficiency of cDNA synthesis was estimated by qRT-PCR using two different primer sets annealing to the 5′ and 3′ ends, respectively, of a control gene (At3g26650, GAPDH, glyceraldehyde-3-phosphate dehydrogenase). Primer sequences were as follows: for GAPDH3′, forward 5′-TTGGTGACAACAGGTCAAGCA-3′ and reverse 5′-AAACTTGTCGCTCAATGCAATC-3′; for GAPDH5′, forward 5′-TCTCGATCTCAATTTCGCAAAA-3′ and reverse 5′-CGAAACCGTTGATTCCGATTC-3′. Transcript levels of each gene were normalized to ACTIN2 (At3g18780) transcript abundance (forward 5′-TCCCTCAGCACATTCCAGCAGAT-3′ and reverse 5′-AACGATTCCTGGACCTGCCTCATC-3′). A total of 187 ROS-responsive genes (Wu et al., 2012) and 1880 TFs (Czechowski et al., 2004; Balazadeh et al., 2008) were analyzed by qRT-PCR as previously described (Caldana et al., 2007; Balazadeh et al., 2008). PCR reactions were run on an ABI PRISM 7900HT sequence detection system (Applied Biosystems, Darmstadt, Germany), and amplification products were visualized using SYBR Green (Applied Biosystems).

Levels of H₂O₂ were determined using the Amplex® Red Technology (Life Technologies, Darmstadt, Germany) following the manufacturer's instructions. Amplex Red (N-acetyl-3,7-dihydroxyphenoxazine) reacts with H₂O₂ in the presence of horseradish peroxidase and forms the fluorescent product resorufin. For the determinations, 100 mg leaves (fresh weight) were ground in liquid nitrogen into a fine powder and resuspended with 0.15 mL extraction buffer prepared as indicated by the manufacturer. This suspension was centrifuged at 4°C at 13,000 rpm for 15 min. Five μL of the supernatant, 45 μL distilled water and 50 μL of Amplex® Red solution were added to a microtitre plate. After 30 min incubation in the dark fluorescence was measured by excitation at 560 nm and emission reads at 590 nm. A calibration curve was established with known H₂O₂ concentrations.

The two genes that were most strongly induced under photorespiratory conditions in GO plants at the 0.5 and 6 h time points (At3g02840 and At1g17180, respectively) were used as baits to identify globally coexpressed genes using the ATTED-II database (http://atted.jp), which allows evaluating genes that are coexpressed under five experimental conditions (tissue, abiotic stress, biotic stress, hormones, and light conditions) (Obayashi et al., 2009).

The production of H₂O₂ in leaves of plants overexpressing GO in the plastids (Fahnenstich et al., 2008) was analyzed after activation of photorespiration by transferring high CO₂-grown plants to ambient-CO₂ conditions. As shown in Table 1, higher levels of H₂O₂ were determined in GO than in WT plants at 0.5 and 4 h after transfer while GO and WT plants maintained under non-photorespiratory conditions (3000 ppm CO₂) showed similar H₂O₂ levels at both time points (Table 1). Note, that as the measurements were performed using whole-leaf extracts the expected differences in chloroplastic H₂O₂ levels between GO and WT plants under photorespiratory condition may be higher than determined here.

To study the impact of an abrupt production of H₂O₂ in chloroplasts on nuclear gene expression, we analyzed transcript level changes of 187 ROS-responsive genes using a previously established qRT-PCR platform (detailed in Wu et al., 2012). The genes included in the platform were chosen from published reports and our own experiments and represent four different groups that were already shown to be rapidly induced by (1) superoxide radical (O⁻₂; 18 genes), (2) singlet oxygen (¹O₂; 22 genes), (3) H₂O₂ (53 genes), or (4) different types of ROS (general ROS-responsive genes; 94 in total).

Gene expression was analyzed in whole rosettes of 3-week-old WT and GO plants at 0.5, 4, 6, and 12 h after shifting high-CO₂-grown plants (non-photorespiratory condition) to ambient CO₂ concentration (photorespiratory condition). Expression profiling was performed in two biological replicates and log-fold change (log2 FC) ratios of expression changes were calculated for GO and WT plants by comparing gene expression levels before and after the CO₂ concentration shift. A total of 131 genes were expressed in all examined samples (Table A1 in Appendix). The remaining 56 genes did not yield detectable PCR amplicons, indicating no or marginal expression under our experimental conditions.

Considering a 3-fold expression difference cut-off, 120 genes displayed differential expression in GO and/or WT plants upon transfer from high to ambient CO₂ concentration; the vast majority of the affected genes (116 in total) were up-regulated, and only four genes were down-regulated (Figure 1, Table A1 in Appendix). Most noticeably, expression of 58 genes was induced in GO plants already within 0.5 h after the transfer to ambient CO₂ condition, whilst only a single gene was induced in the wild type at the same time point (Figure 1). Importantly, however, many genes showed also high expression in the wild type at later time points after the CO₂ concentration shift, but the expressional changes were in most cases more pronounced in GO than WT plants (Figure 1, and section “Early Induction of Indole Glucosinolate and Camalexin Biosynthesis Genes in GO Plants”). Thus, our data indicate that similar sets of ROS-responsive genes responded to the CO₂ shift in GO and WT plants; however, the dynamics of the transcriptional responses were clearly different in the two types of plants, being faster and more prominent in the GO plants.

To identify transcripts responsive to metabolically produced H₂O₂ we focused our analysis on the 0.5- and 6-h time points. Genes were considered differentially expressed when the fold change was more than 3-fold (log₂ ≥ 1.56).

At 0.5 h after shifting plants to ambient CO₂ concentration, 58 of the 131 expressed genes were induced in GO plants by more than 3-fold, whilst in the wild type the expression change was less than 3-fold, suggesting that these genes participate in early signaling steps triggered by the production of H₂O₂ under photorespiratory conditions (Table 2). After 6 h, seven of these genes showed WT levels of expression (below 3-fold), while 29 were further overexpressed only in GO (Table 2). Although at 6 h after transfer to ambient CO₂ the expression fold-change (FC) of the remaining 22 genes was higher than 3 in both, GO and WT plants, the expression change between GO and WT (FC_GO/FC_WT) was higher than 2 for 16 of these genes (Table 2), indicating that their higher expression in GO plants was triggered by the elevated levels of H₂O₂.

Later responding genes, which were affected only after 6 h under photorespiratory conditions, were also identified. From the 23 genes that showed an expression change of above 3-fold in GO, 13 were only induced in GO, while 10 genes were induced in both, GO and WT. The FC ratio in GO and WT (FC_GO/FC_WT) was above 2 for the 10 genes (Table 3), indicating that their expression in GO plants is controlled by the higher levels of H₂O₂, similar to the early-responsive genes.

The most highly up-regulated gene in GO plants at 0.5 h after induction of H₂O₂ production was At3g02840 (encoding a putative U-box-type E3 ubiquitin ligase, known to respond immediately-early to fungal elicitation) (Table 2). We used the ATTED-II database (http://atted.jp; Obayashi et al., 2009) to discover genes coexpressed with At3g02840 and observed that 45 of the 58 genes induced at 0.5 h after induction of H₂O₂ production cluster together (Table 2), indicating that metabolically produced H₂O₂ in GO plants induces the coordinate expression of functionally related genes. A similar analysis using the most highly expressed gene at 6 h after induction of H₂O₂ production (At1g17180, encoding glutathione S-transferase Tau 25) indicated that another group of eight genes are coordinately expressed in GO plants at this later time point (Table 3).

Recently, Inzé et al. (2012) listed the 85 most strongly H₂O₂-responsive genes in catalase loss-of-function mutants shifted from low- to high-light conditions, where H₂O₂ is produced in peroxisomes by the action of photorespiratory GOs. Interestingly, 23 of the 81 genes, which changed their expression in the GO plants were also differentially expressed in catalase loss-of-function mutants (Tables 2 and 3), indicating that they respond to enhanced levels of H₂O₂ independent of the site of its generation; the remaining genes may then represent candidates preferentially responsive to H₂O₂ produced in chloroplasts. Many of the genes up-regulated in GO plants encode proteins or TFs of currently unknown specific functions. Interestingly, however, several of the early-responsive genes are involved in tryptophan-derived biosynthesis of the phytoanticipins camalexin and indole glucosinolates, i.e., secondary metabolites that have antifungal and insect-deterring functions (Kliebenstein et al., 2001; Bednarek et al., 2009). These genes encode (1) the transcription factor WRKY33 (At2g38470), which is involved in controlling camalexin biosynthesis (Birkenbihl et al., 2012); (2) the Myb-type transcription factor HIG1/MYB51 (At1g18570) involved in the positive regulation of indole glucosinolate biosynthesis by activating several target genes (Gigolashvili et al., 2007); (3) the O-methyltransferases IGMT1 (At1g21100) and IGMT2 (At1g21120), which catalyze the transfer of a methyl group to the hydroxy indole glucosinolate hydroxyindol-3-ylmethylglucosinolate (4 and 1OH-I3M, respectively) to form methoxyindol-3-ylmethylglucosinolate (4 and 1MO-I3M, respectively) (Pfalz et al., 2011); and (4) cytochrome P450 monooxygenase CYP81F2 (At5g57220), that is essential for the pathogen-induced accumulation of 4-methoxyindol-3-ylmethylglucosinolate (4MI3G) (Bednarek et al., 2009). Our data thus show the early induction of indole glucosinolate and camalexin biosynthesis genes in GO plants after metabolic formation of H₂O₂ through the activation of genes encoding enzymes involved in intermediate metabolite conversions and of TFs that act on several target genes of these biosynthetic pathways.

To understand the potential effects of overexpression of GO in chloroplasts on the nuclear transcriptional program, we next broadened our analysis by testing the expression of 1880 TFs using a highly sensitive quantitative real-time PCR (qRT-PCR) platform (Czechowski et al., 2004; Balazadeh et al., 2008). Considering the data obtained from the profiling of the ROS-responsive genes, we analyzed the expression at 0.5 h after induction of H₂O₂ production to capture the early-responsive TFs. Expression profiling was performed in two biological replicates and log-fold change (log2 FC) ratios of expression changes were calculated for GO and WT plants by comparing gene expression levels before and after the transfer of plants grown at high CO₂ to ambient CO₂.

TFs most strongly responding to H₂O₂ were identified by comparing their expression FC in GO and WT plants. A TF was considered differentially expressed when the FC in GO was more than 3-fold (log₂ ≥ 1.56) and less than 2-fold in the wild type (log₂ ≥ 1.0) (Table 4). Analysis of transcript profiles revealed that the expression of 1449 genes, representing 77% of all TF genes tested, could be detected (Table A2 in Appendix). The remaining 23% (431 of the 1880 TFs) did not yield detectable PCR amplicons, indicating no or very weak expression in the tested material.

At 0.5 h after shifting plants to ambient CO₂ concentration, 78 of the 1449 genes were induced by more than 3-fold in GO plants, whereas in WT plants the expression changes of the same genes were less than 2-fold (Table 4). Using published data the involvement/participation of the TFs in specific biological processes (Table 4) could be assessed, which allowed the classification of the TFs into five functional groups (FG) enriched with specific gene ontology categories (Figure 2). FG1 contains TFs involved in the regulation of proanthocyanidin and anthocyanin biosynthesis (Table 4 and Figure 2). The TFs TT8 and MYB75 affecting the gene expression of dihydroflavonol 4-reductase (Debeaujon et al., 2003) are included in this FG. FG2 contains TFs affecting developmental processes like lateral root formation (GATA23), flowering (FD1, ANAC089, TEM2 and SNZ), shoot branching (MYB2 and BRC2), senescence (ANAC092/ORE1) and cell division (ANAC068 and HAT4) (Table 4 and Figure 2). The activation of these TFs in GO plants would result in altered growth and flowering (see below and Fahnenstich et al., 2008). FG3 includes TFs and TF-interacting proteins negatively regulating jasmonate (JA) signaling (JAZ7, JAZ8, JAZ9, JAZ10, WRKY50, and WRKY51; Chico et al., 2008; Staswick, 2009; Gao et al., 2011) (Table 4 and Figure 2). JAZ proteins bind directly to the key transcription factor MYC2 and thereby prevent JA-dependent gene transcription (Chini et al., 2007; Pauwels et al., 2010). At the same time JAZ genes are rapidly induced by JA and some are MYC2-regulated. This feedback loop regulation would provide a rapid on and off switch of the pathway involving JA. Transcriptional activation of JAZ genes was found to occur in response to several biotic and abiotic challenges (Yan et al., 2007). JAZ proteins would also exert their effects on post-wound inhibition of vegetative growth in A. thaliana (Yan et al., 2007) and as repressors of necrosis and/or programmed cell death during development in tobacco (Oh et al., 2012). In GO plants, the action of JAZ genes together with those of FG2 would impact growth and reproductive capacity, resulting in altered development under conditions that promote the formation of H₂O₂. FG4 includes TFs with diverse functions in plant defense and signaling, e.g., activators of tryptophan-derived biosynthesis of camalexin (JUB1/ANAC042) and indole glucosinolates (MYB122), as well as regulators of photomorphogenesis (STH2) (Table 4 and Figure 2). The early activation of camalexin and indole glucosinolate biosynthesis was also observed in the analysis performed with the ROS-responsive gene platform (Table 1). Finally, FG5 includes TFs with currently unknown functions (Table 4 and Figure 2).

The analysis of the transcript profiles at 0.5 h after induction of H₂O₂ production in GO plants (Table A2 in Appendix) also revealed a group of 13 genes that are down-regulated in GO relative to WT plants (Table 5). The function of five of these genes is currently unknown, but interestingly, the remaining eight genes positively control developmental processes. The down-regulation of expression of these TFs in GO plants together with the up-regulation of expression of TFs negatively affecting development (see FG2, Table 3) would act in concert to arrest growth and especially to delay the transition from the vegetative to the reproductive phase. Consistently, our previous results showed that GO plants growing under photorespiratory conditions are smaller than WT plants, presenting a reduced rosette diameter and a delay in flowering time (Fahnenstich et al., 2008).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.