Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS

Michael Wrzaczek; Mikael Brosché; Hannes Kollist; Jaakko Kangasjärvi

doi:10.1073/pnas.0808980106

New Research In

Edited by Joseph R. Ecker, The Salk Institute, La Jolla, CA, and approved February 2, 2009 (received for review September 9, 2008)

Abstract

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide- and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development.

Keywords:

Reactive oxygen species (ROS) have earlier been considered merely as cytotoxic compounds, but clearly, their functions are far more diverse. They have important and tightly regulated roles as signaling molecules in disease resistance, stress adaptation, and development in many organisms (1, 2). In plants, ROS are produced in response to many stresses, including pathogen attack, osmotic stress, excess light, wounding, and ozone (O₃), in different subcellular compartments (1). For example, pathogen attack and O₃ induce an oxidative burst in the extracellular space, whereas high light or the herbicide methyl viologen lead to the production of ROS in the chloroplast (1). ROS also serve as second messengers in abscisic acid (ABA) signaling in the regulation of the stomatal aperture (3) and are involved in root hair growth (4). In angiosperm flowers, ROS are present in pollen and stigmas (5) and regulate pollen tube growth (6), but they might also have further functions including self-incompatibility and pathogen defense (5).

Cells decipher ROS signals with regard to type, localization, and timing of ROS produced (7), but mechanisms of ROS perception have remained mostly obscure. Only a few examples have linked the cellular machinery to perception and integration of ROS signals. Plant heat-shock transcription factors (HSFs) might function as H₂O₂ sensors (8), and the salicylic acid (SA) signaling protein NPR1 confers redox regulation to the transcription factor TGA1 (9). Various components of the ROS detoxification machinery could be used to detect differences in the oxidative load (9, 10). However, the mechanisms and processes involved in the ROS perception and signaling in the extracellular space have remained almost completely elusive.

Programmed cell death (PCD) is a common response to various environmental and developmental cues (7). In plant–pathogen interactions, the hypersensitive response (HR) protects plants against a biotrophic pathogen by isolating it in a patch of dead tissue. Limitation of cell death is crucial because extended tissue loss would have detrimental effects. In extreme cases, the inability to regulate lesion spread can lead to the death of the plant (7, 11, 12). ROS are centrally involved in the regulation of PCD (7), but the precise roles in lesion initiation, spread, and containment are not well characterized. Potentially, ROS can function as both positive and negative regulators of PCD, depending on conditions and requirements (7, 12, 13).

In this study, we characterize a component involved in the regulation of stress-induced cell death in plants. The GRIM REAPER (GRI) protein is an Arabidopsis ortholog of the tobacco stigma-specific protein 1 (STIG1) (14). STIG1 helps to regulate exudate secretion in the pistils of petunia and tobacco (15). Tomato LeSTIG1 binds to the extracellular domain of pollen receptor kinases and promotes pollen tube growth in vitro (16). However, no roles for STIG1 have been described in vegetative tissues or in the regulation of cell death. Here, we present evidence that GRI is involved in the regulation of ROS-induced cell death in Arabidopsis leaves in a superoxide- and SA-dependent manner.

Results

Isolation and Identification of an Ozone-Sensitive Arabidopsis thaliana Mutant.

A reverse genetic screen for ozone sensitivity was performed in Arabidopsis with ≈80 T-DNA and transposon lines bearing insertions in O₃-regulated genes [supporting information (SI) Table S1). Plants were exposed to 300 parts per billion (ppb) O₃ for 6 h. One O₃-sensitive line was a dSPM insertion mutant (SM_3.39219) in the locus At1g53130; leaves of the O₃-exposed plants exhibited HR-like lesions of collapsed tissue (Fig. 1A). The mutant was designated grim reaper (gri). Cell death, quantified as ion leakage, was significantly increased in gri when compared with clean-air controls and O₃-exposed wild-type Col-0 plants (Fig. 1B). Plant O₃-sensitivity can be a result of altered stomatal function with increased O₃ entry to the leaves (3). In gri, stomatal regulation was not impaired (Fig. S1A), and O₃ uptake to the leaf was the same as in Col-0 (Fig. S1B), suggesting that O₃ sensitivity of gri is caused by impaired sensing of, or response to, O₃ or ROS derived from O₃ by mesophyll cells.

Fig. 1.

gri plants show increased ROS-induced cell death and reduced seed content. (A) Col-0 and gri plants were exposed to 300 ppb O₃ for 6 h. gri plants showed lesions after O₃ treatment, whereas no damage was visible in Col-0. (B) Ion leakage was determined 8 h after the onset of O₃ exposure. In gri, but not in Col-0 and vector control (VC), ion leakage increased after exposure to O₃. In gri plants carrying a genomic complementation construct (gGri) and plants overexpressing GRI-c-myc (OE), ion leakage increased after O₃ treatment compared with Col-0, however, to lesser levels than in gri. The experiment was repeated 5 times with similar results. Four genomic complementation and overexpression lines were tested, and representative results from 1 line are shown. (C) The seed content in siliques was reduced in gri and GRI overexpressors compared with Col-0 and vector control. (D) GRI protein possesses a signal peptide and a STIG1-domain. The dSPM insertion site in gri and the location of primers used for RT-PCR experiments are shown. (E) RT-PCR analysis showed that the full-length GRI transcript was absent from gri plants. Primers 1 and 2 show the transcript before the insertion in Col-0 and gri. Primers 3 and 4 show a transcript in Col-0 but not in gri after the insertion site. Actin-2 transcript is present in Col-0 and gri plants. In B and C, all data points are mean ± SD (in B, n = 4; in C, n = 6). Bars labeled with a different letter differ significantly (P < 0.05) by Tukey's honestly significant difference (HSD) test.

GRI Encodes a Small Protein with Predicted Extracellular Localization.

The intronless GRI gene encodes a 18.6-kDa protein of 169 aa with a STIG1 domain (PF04885, amino acids 33–168) and a predicted N-terminal signal peptide (amino acids 1–30) for the secretory pathway (Fig. S2A). The STIG1 domain indicates that GRI is related to tobacco and petunia stigma-specific protein, STIG1 (14, 15). The tobacco Stig1 encodes a small secreted protein present in the stigmatic lipid exudates. In Arabidopsis, GRI belongs to a small gene family of 6 members (Fig. S2B). Neighbor-joining and distance analyses point to GRI as the Arabidopsis ortholog of tobacco STIG1 (Fig. S2 B and C).

GRI Expression Is Very Low in Leaves and High in Flowers.

The GRI transcript was present in leaves at very low levels and showed a slight circadian variation in its expression (Table S2). In flowers, GRI expression was 1,000-fold higher than in leaves. Despite the very low expression of GRI in leaves, a clear O₃-sensitive leaf phenotype was visible in gri (Fig. 1A) and exposure to 300 ppb O₃ for 6 h increased GRI transcript abundance in leaves ≈2- to 3-fold.

Seed Content Is Reduced in gri Siliques.

The only phenotype observed after silencing of tobacco Stig1 was an acceleration of secretion of the stigmatic exudate and a slightly different appearance of the stigma (15). In gri, no apparent changes of flower morphology were found, but the seed content of the siliques was reduced in gri and GRI-c-myc/StrepII overxpression plants compared with Col-0 (Fig. 1C).

Infiltration of Arabidopsis Leaves with GRI-Peptide Induces Cell Death.

In genetic complementation assays, the O₃-sensitive phenotype of gri was not fully complemented when gri plants were transformed with the genomic clone of GRI (Fig. 1B). In addition, Col-0 plants overexpressing the GRI ORF with a C-terminal c-myc/StrepII tag under the control of the cauliflower mosaic virus 35S promoter showed a similar O₃-sensitive phenotype as gri. Ion leakage after O₃ treatment was not as strongly increased in the complementation and overexpression lines as in the gri mutant, but the levels were clearly increased compared with Col-0 (Fig. 1B). The location of the dSPM insertion in gri (Fig. 1D) suggested that a N-terminal part of the GRI protein (96 aa including the signal sequence) might still be produced in the gri mutant. Indeed, RT-PCR analysis indicated that GRI transcript abundance was similar in Col-0 and gri upstream of the insertion site, whereas downstream of the transposon insertion, the GRI transcript was not detectable by PCR in gri (Fig. 1E). This implied that gri plants could express a truncated GRI protein that could cause the observed phenotypes. Alternatively, the observed phenotypes might be due to partial cosuppression of the GRI gene.

When the GRI protein with C-terminal c-myc/StrepII tag was expressed in Col-0, in addition to the ≈35-kDa GRI-c-myc/StrepII fusion protein, a smaller, ≈25-kDa protein was recognized by the α-c-myc antibody (Fig. 2A). This implies that an ≈10-kDa fragment is released from the N terminus of the fusion protein. Because of the predicted size of the GRI signal peptide (30 aa, ≈3.5 kDa), the released N-terminal fragment would contain ≈60–70 additional amino acids of the GRI protein. A peptide predicted to be present in the gri mutant, corresponding to amino acids 31–96 after the signal peptide sequence (GRI-peptide), was targeted for further study.

Fig. 2.

A short GRI peptide is capable of inducing cell death in a superoxide-dependent manner. (A) GRI-c-myc/StrepII protein is present in plants overexpressing GRI. Western blot analysis with α-c-myc antibody detects a 35- and 26-kDa GRI-C-terminal c-myc/StrepII fusion protein. Both proteins are absent in Col-0, gri, and vector control (VC). Equal loading was shown by amido black staining. (B) Infiltration of GRI-peptide into Col-0 leaves induced cell death. (C) Coinfiltration of GRI-peptide with SOD and CAT reduced cell death. (D) Cell death induction by GRI-peptide is reduced in atrbohD, atrbohD/F, and sid2. However, in atrbohF, cell death is induced similarly to Col-0. (E) X/XO infiltration into leaves induced more cell death in gri and OE plants than in Col-0. In B–E data points are mean ± SD (n = 6), differences larger than LSD shown are statistically significant (P < 0.05) by the modified least-significant difference test. (F) Growth of virulent Pst DC3000 was reduced in gri compared with WT, whereas the growth of an avirulent variant (Pst DC3000 avrRpt2+) was not affected. Data points are mean ± SD (n = 3). Bars labeled with different letters differ significantly (P < 0.05) by Tukey's HSD test.

GRI-peptide was produced in Escherichia coli and purified by using a GST tag. The recombinant protein was infiltrated into Col-0 leaves, and cell death was quantified as ion leakage. Infiltration with the GST-GRI-peptide or the GRI-peptide without the GST-tag induced ion leakage, whereas bacterially produced GST or buffer alone had no such effect (Fig. 2B). This offers an explanation for the incomplete rescue of the gri mutant by genomic complementation and also for the O₃-sensitive phenotype of the plants overexpressing the GRI-c-myc/StrepII fusion protein. The presence of a truncated protein in gri plants, corresponding to GRI-peptide, can cause cell death under conditions of extracellular oxidative stress, which is apparent as increased sensitivity to O₃.

GRI-Peptide-Induced Cell Death Is Superoxide Dependent.

Cell death was increased in gri plants only under stress conditions. To test whether ROS were required for the induction of cell death, we included ROS scavengers in the infiltration buffer together with GRI-peptide. The removal of superoxide through coinfiltration with superoxide dismutase (SOD) reduced GRI peptide-induced cell death to control levels (Fig. 2C). Catalase (CAT), a H₂O₂ scavenger, also slightly reduced ion leakage. GRI-peptide did not cause increased cell death in atrbohD, a mutant in the plant respiratory burst NADPH oxidase generating superoxide, but in atrbohF, cell death was induced to a similarly to Col-0 (Fig. 2D). Induction of cell death in atrbohD/F was similar as in atrbohD. GRI-peptide was not able to induce cell death in sid2, impaired in SA biosynthesis, whereas a weaker reduction in the level of GRI-peptide-induced cell death was observed in triple fad, impaired in jasmonic acid (JA) biosynthesis, and in ein2, impaired in ethylene signaling (Fig. S3). Infiltration of leaves with the exogenous superoxide producing system xanthine and xanthine oxidase (X/XO), which enzymatically generates superoxide in the extracellular space (14), caused increased cell death in the gri mutant (Fig. 2E). The gri plants also accumulated substantial amounts of superoxide after exposure to O₃ (Fig. S4). These results together demonstrate that the cell death-inducing effect of GRI-peptide depends on the presence of superoxide and SA.

gri Is More Resistant to a Virulent Bacterial Pathogen.

O₃-induced cell death shares similarities with the pathogen-induced HR (1, 7). Both events feature an extracellular oxidative burst, which is involved in the regulation of hypersensitive cell death and other defense pathways (1). Thus, altered sensitivity to ROS in cell death initiation in gri could have an impact on plant resistance to pathogens. We addressed this in growth assays with the virulent pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) and avirulent DC3000 avrRpt2+. gri plants showed increased resistance to the virulent strain, but no change in the growth of the avirulent strain (Fig. 2F). Cell death progressed at a faster rate in response to both virulent and avirulent strains in the gri mutant compared with control plants (Fig. S5). Taken together, these results suggest that increased cell death in gri might lead to the increased resistance to a virulent bacterial pathogen and faster induction of cell death to avirulent pathogens.

Hormone Responses Are Suppressed in the gri Mutant.

Several hormones are involved in plant stress responses, notably SA, JA, and ethylene (7). To investigate whether signaling pathways involving these 3 hormones were disturbed in gri, we analyzed the expression of marker genes for these hormones (Table 1). Upon O₃ exposure ICS1, encoding a protein required for SA biosynthesis, was induced strongly in Col-0, but weakly in gri. The induction of downstream SA target genes, PR1 and a putative ABC transporter At1g15520, was similarly reduced in gri. JA marker genes LOX4 and MDHAR (monodehydroascorbate reductase) were induced by O₃ in Col-0, but in gri this induction was mostly absent. Another JA defense marker, PDF1.2, showed an earlier induction in gri compared with Col-0, but at later time points the transcript did not accumulate to the same levels as in Col-0. The induction of ACS6, encoding the rate-limiting enzyme in O₃-induced ethylene biosynthesis, was not altered in gri after O₃ exposure when compared with Col-0. In summary, this shows that responses leading to the initiation of SA- and JA-related processes are disturbed in gri, whereas ethylene-related processes remain unaffected.

View this table:

Table 1.

Hormone signaling is impaired in gri

Altered marker gene expression could be a result of altered hormone accumulation; thus, we analyzed hormone concentrations after O₃ exposure (Fig. 3). Concentrations of free SA and JA under control conditions were not significantly different between Col-0 and gri. No induction of SA and JA was observed at 2 h after the onset of O₃ treatment. However, free SA and JA accumulated to lower levels in gri than Col-0 8 h after onset of the O₃ treatment. The concentration of ABA, another plant hormone implicated in the responses to various stresses (17) did not differ between Col-0 and gri after O₃ exposure (Fig. S6). These results indicated that SA and JA accumulation, typical for O₃-induced cell death, was strongly reduced in gri.

Fig. 3.

Salicylic acid and jasmonic acid induction is reduced in gri. Plants were exposed to 300 ppb O₃ for 6 h. Samples were taken at 0, 2, 8, and 24 h after onset of ozone exposure from exposed and clean air control plants. The concentrations of plant hormones free salicylic acid (SA) (A) and jasmonic acid (JA) (B) were measured. Data points are mean ± SD (n = 3). Bars labeled with different letters differ significantly (P < 0.05) by Tukey's HSD test.

GRI Protein Is Secreted to the Extracellular Space.

The presence of an N-terminal signal peptide for secretion indicates that the GRI protein could be secreted to the extracellular space. To test this, the GRI protein-coding sequence (amino acids 1–169) was fused in-frame with yellow fluorescent protein (YFP). In addition, the GRI-coding sequence lacking the N-terminal signal peptide (amino acids 31–169) and the GRI signal peptide alone (amino acids 1–30) were fused with YFP. Onion epidermal cells were transiently transformed with the constructs via particle bombardment and the subcellular localization of the protein-YFP fusions was examined by confocal microscopy (Fig. 4). YFP alone (Fig. 4 A–D) and GRI (31–169)-YFP (Fig. S7 E–H) missing the N-terminal signal localized to cytoplasm and nucleus. After plasmolysis, fluorescence was visible only in cytoplasm detached from the cell wall and in the nucleus. In cells expressing GRI (1–30)-YFP and GRI (1–169)-YFP, the fusion protein was detected in the cytoplasm. However, in plasmolyzed cells, the fusion protein was also visible in the extracellular space in regions where the plasma membrane detached from the cell wall (Fig. 4 E–H and Fig. S7 A–D, indicated with arrows). Under similar conditions the SLAC1-YFP fusion protein, which localizes to the plasma membrane (3), did not display any signal from the cell wall (Fig. S7 I–K). Taken together, the results indicate that GRI (1–169)-YFP and GRI (1–30)-YFP fusion proteins were secreted to the extracellular space.

Fig. 4.

GRI-signal peptide-YFP localizes to the cytoplasm and extracellular space of transformed onion cells. Onion epidermis was bombarded with DNA-coated gold particles to examine the expression of different constructs. (A and B) YFP expression in onion epidermis cells, YFP and YFP-transmission light overlay are shown. (C and D) After plasmolysis with 1 M NaCl for 20 min, the cytoplasm detached from the cell walls. (E and F) YFP localizes to the nucleus and cytoplasm. GRI-signal peptide-YFP localized in the cytoplasm and perinuclear region of the cells. (G and H) After plasmolysis the recombinant protein was detectable in the cytoplasm but also remaining in the cell wall where plasma membrane and cytoplasm have detached (arrows).

Discussion

Here, we present evidence for the involvement of Arabidopsis GRIM REAPER (GRI) protein in the positive regulation of ROS-induced cell death. The GRI protein is related to tobacco STIG1, which is predominantly present in tobacco stigma and required for stigmatic lipid exudate secretion (15). The tomato ortholog, LeSTIG1, binds the extracellular domain of 2 pollen receptor kinases in vitro (16). Consistent with a role for STIG1 in flowers, GRI transcript abundance is very low in Arabidopsis leaves and significantly higher in flowers (Table S2). Unlike tobacco, Arabidopsis stigma do not secrete lipid exudates, which makes comparisons between tobacco STIG1-antisense plants and gri difficult. However, whereas gri flowers looked normal, the seed content in gri siliques was reduced. Furthermore, gri crosses had severely reduced efficiency, supported by the reduction of the seed number of siliques (Fig. 1C), suggesting that GRI also has a function in Arabidopsis flowers.

A function for STIG1 has been shown in tobacco and petunia flowers, but no connection to vegetative tissues or cell death has been implicated. The O₃-sensitive phenotype of the Arabidopsis gri mutant, hallmarked by lesion development and increased ion leakage after O₃ exposure, demonstrates important cell death- and ROS-related functions of GRI in vegetative tissues. The O₃-sensitive phenotype of the GRI-overexpression plants (Fig. 1B), the incomplete complementation of gri by a genomic clone (Fig. 1B), as well as the apparent release of an N-terminal fragment (Fig. 2A), implies a gain-of-function model for the truncated GRI protein. RT-PCR analysis showed that a 5′ part of the GRI transcript was still present in gri and, thus a truncated GRI protein, similar in size to the fragment that is released from the N terminus of GRI, could be produced. Infiltration of a recombinant peptide corresponding to this truncated protein induced cell death in Arabidopsis leaves. This suggests that the GRI N-terminal part is involved in the regulation of cell death. This, however, poses the question of how gri plants survive under normal growth conditions in the presence of a potentially lethal truncated GRI protein. Our results point out that GRI-peptide needs an additional signal for the cell death induction. However, it is also possible that gri plants, facing the challenge of “living” in the presence of the peptide, might survive through up-regulation of negative regulators of cell death.

ROS are involved in the regulation of various types of cell death (7). Removal of superoxide by coinfiltration of the GRI-peptide with SOD reduced cell death to control levels. A weaker reduction was observed by H₂O₂ removal with catalase. Plant respiratory burst oxidases (Atrboh) are proposed to be a major source of extracellular superoxide. AtrbohD and AtrbohF are the highest expressed members in leaves (13). Interestingly, cell death induced by GRI peptide was eliminated in atrbohD (and the double atrbohD/F) but not in atrbohF. This suggests that superoxide derived from AtrbohD is required for GRI peptide-induced cell death. The differential response of atrbohD and atrbohF is consistent with earlier results showing different functions for these mutants; atrbohD lacked most ROS production in response to a fungal pathogen whereas atrbohF allowed for enhanced cell death and improved pathogen resistance (13). The role of superoxide in cell death initiation by GRI-peptide is further supported by the increased sensitivity of gri to X/XO, as well as the increased resistance to a virulent bacterial pathogen. The superoxide produced upon pathogen infection triggers cell death, which is enhanced by the truncated GRI protein, subsequently leading to pathogen resistance. This makes GRI-peptide a potential candidate for being involved in ROS-induced cell death. This role of the N-terminal GRI-peptide could explain the phenotype of gri plants as well as the phenotypes of the GRI overexpression lines and the genomic complementation lines. Developmental cell death has a clear role in plant reproductive biology (18). Given the abundance of ROS in floral organs and their involvement in pollen tube growth (6), the presence of a truncated GRI peptide in gri plants offers also an explanation for the reduced seed content in gri siliques as preactivated GRI-peptide in the presence of ROS may lead to misregulated developmental cell death.

Infiltration of GRI-peptide into sid2, impaired in SA production, abolished the induction of cell death. This suggested that cell death caused by infiltration of GRI-peptide depends on SA. However, O₃ induction of SA accumulation and expression of SA marker genes were reduced in gri. This implied that gri might be preprimed for the execution of cell death, or have altered sensitivity to SA, and only need a low increase in SA levels to execute the cell death program.

The presence of a signal peptide for the secretory pathway and induction of cell death by infiltration of GRI-peptide into the extracellular space of Arabidopsis leaves points to a function of GRI in the extracellular space. This is supported by secretion of STIG1 into the stigmatic lipid exudate (15) and binding of LeSTIG1 to the extracellular domain of RLKs (16). Because of the very weak expression of GRI in Arabidopsis leaves, analysis of the subcellular localization of GRI required overexpression with the 35S promoter. Microscopic analysis of GRI-YFP localization suggested both cytosolic and extracellular localization for GRI-YFP. Similarly, when fused to YFP [GRI(1–30)-YFP], the GRI signal peptide directed YFP to the cell wall. Localization of the GRI-YFP fusion protein to the cytoplasm might be due to mislocalization caused by the 35S promoter, or an additional stimulus or protein might be required for correct GRI localization (19). Furthermore, fluorescent proteins frequently become unstable in the extracellular environment, accounting for very low fluorescence levels (19). Further experiments will be required for determining the precise subcellular localization under control and stress conditions using an inducible promoter system and a transgenic approach in Arabidopsis. Taken together, our results provide evidence that the GRI protein is secreted to the extracellular space and can perceive signals there.

The O₃-sensitive phenotype of gri can serve as a model for ROS regulation of cell death under stress conditions and potentially also during flower development. The gri mutant shows a way to short-circuit the cell death regulation leading to increased cell death under conditions of oxidative stress in the extracellular space. Our results suggest that GRI is involved in mediating the effects of extracellular superoxide for the regulation of cell death. The presence of truncated GRI peptide is sufficient for the initiation of ROS-induced cell death. It will be interesting to analyze the mechanisms that regulate this property of GRI protein in the future.

Materials and Methods

Plant Material and Growth Conditions.

The dSPM insertion line SM_3.39219 (N125930) was obtained from the Nottingham Arabidopsis Stock Centre (http://nasc.nott.ac.uk) together with other insertion lines (listed in Table S1). Presence of the insertion and homozygous plants were verified by PCR using gene-specific primers (SI Text). Stomatal conductance was analyzed as previously described (20). sid2-1 was obtained from Jean-Pierre Métraux (University of Fribourg, Fribourg, Switzerland), triple fad was obtained from John Browse (Washington State University, Pullman, WA), and atrbohD, atrbohF and atrbohD/F were obtained from Miguel Torres (Universidad Politecnica de Madrid, Spain).

Ozone Treatments.

Twenty-one-day-old Arabidopsis plants were exposed to 300 ppb O₃ for 6 h. Samples were harvested at the indicated times after the onset of the ozone treatment. Cell death was quantified by ion leakage as previously described (21).

Quantitative PCR Analysis.

RNA was isolated as described (22). Total RNA (5 μg) was DNaseI-treated (Fermentas) and used for cDNA synthesis with SuperScript III (Invitrogen) and Ribolock RNase Inhibitor (Fermentas) according to manufacturers' instructions. The reaction was diluted to a final volume of 50 μL, and 1 μL of cDNA was used as template for PCR by using LightCycler 480 SYBR green I master (Roche Diagnostics) on a LightCycler 480 (Roche Diagnostics) in triplicate. The raw threshold cycle (Ct) values were normalized to Actin-2 and expression ratios calculated by the 2^−ΔΔCt method. Primer sequences are available in SI Text.

Vapor-Phase Hormone Extraction.

The plant hormones SA, JA, and ABA were extracted according to an adapted protocol (23). Measurement by gas chromatography (Thermo Finnigan), and quantification was carried out according to manufacturer's instructions.

Superoxide Treatments.

Extracellular superoxide [xanthine/xanthine oxidase in sodium phosphate buffer, 10 mM (pH7)] was vacuum infiltrated into completely expanded detached leaves from 5-week-old plants as previously described (21, 22). Cell death was measured at indicated times with a Conductivity Meter (Mettler Toledo). Nitroblue tetrazolium (NBT) staining was carried out as previously described (22).

Genetic Complementation and Generation of GRI-c-myc/StrepII-Overexpression Plants.

For genetic complementation the GRI locus including the promoter and the 3′ untranslated region were amplified by using specific primers (SI Text) and cloned into the binary plant expression vector pGreenII0029 (24). Homozygous single-insertion plants were selected by segregation analysis.

Full-length or truncated versions of the GRI cDNA were amplified by using specific primers and sequenced. They were cloned into pGreenII0029 under control of the Cauliflower Mosaic Virus 35S promotor, tagged with c-myc/StrepII or YFP, respectively, and transformed into Col-0 by floral dipping (25). For the construction of GST-GRI-peptide, the corresponding part of the cDNA was amplified by using specific primers and cloned into the bacterial expression vector pGEX-4T-1 (GE Healthcare Lifesciences) and subsequently sequenced.

Protein Extraction and Western Blot Analysis.

Total protein was extracted from leaves by grinding in homogenization buffer (SI Text), followed by centrifugation. Protein amounts in the supernatant were determined by the Bio-Rad Protein Assay according to the manufacturer's specifications. For Western blot analysis, equal amounts of proteins were separated by 12% SDS/PAGE and blotted to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were probed with α-c-myc antibodies (A-14; Santa-Cruz). Horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) was used as a secondary antibody. The reaction was detected by enhanced chemoluminescence by using the Supersignal Pico detection kit (Thermo Scientific).

Peptide Infiltration.

GST-GRI-peptide was produced in E. coli BL21-RIL Codon plus cells (Stratagene) and purified by using Glutathione Sepharose 4B (GE Healthcare Lifesciences) according to manufacturer's instructions. The GST-tag was removed by using thrombin (GE Healthcare Lifesciences) according to manufacturer's instructions. Proteins were quantified by using the Bio-Rad Protein Assay.

GRI-peptide, with or without CAT (Roche) or SOD (Sigma), was diluted to a final concentration of 100 ng/mL in 10 mM sodium phosphate buffer (pH7) and infiltrated into fully expanded leaves of 5-week-old plants. Four leaf disks were cut out, rinsed, and pooled in 5 mL of MilliQ water. Ion leakage was measured by using a conductivity meter.

P. syringae Growth Curve Assay.

P. syringae pv. tomato DC3000 (Pst) or Pst DC3000 avrRpt2+ was used in all experiments (26). Bacteria were grown on NYG medium (0.5% trypton peptone; 0.3% yeast extract; 2% glycerol) plus 1.5% bacto-agar at 28 °C. For plant infection, liquid cultures were inoculated from plates and grown in NYG medium at 28 °C overnight. Plants were vacuum-infiltrated, and bacterial growth was assessed as described (26). Ion leakage was measured as described above.

Onion Transformation by Particle Bombardment.

DNA-coated gold particles (0.6 μm; Bio-Rad) were prepared according to manufacturer's instructions. Onion epidermal cells were transformed via bombardment with a Helios Gene Gun (Bio-Rad) according to manufacturer's instructions. After transformation, the epidermis was floated on 20 mL of 0.5× MS medium in the dark overnight. YFP expression was analyzed with a Leica TCS SP5 confocal microscope. Plasmolysis was carried out after overnight incubation immediately before microscopic analysis by immersing the epidermal peels in 0.5× MS medium with 1 M NaCl for 20 min as previously described (3).

Phylogenetic and Statistical Analysis.

Sequence alignments were performed by using the Clustal W program (27). Neighbor-joining trees and matrices were constructed by using the Mega 4 software package (28). One hundred bootstrap sets were used.

Acknowledgments

We thank Pinja Jaspers, Kirk Overmyer and Jorma Vahala for comments on the manuscript, Günter Brader for help with hormone measurements and Triin Vahisalu with screening T-DNA lines. The c-myc and YFP tags were a gift from Claudia Jonak (Gregor Mendel Institute Vienna). The project was financed by University of Helsinki and the Academy of Finland Centre of Excellence program 2006–2011. HK was financed by Estonian Science Foundation and Ministry of Science and Education.

Footnotes

¹To whom correspondence should be addressed. E-mail: jaakko.kangasjarvi{at}helsinki.fi
Author contributions: M.W., M.B., and J.K. designed research; M.W., M.B., and H.K. performed research; M.W., M.B., H.K., and J.K. analyzed data; and M.W., M.B., and J.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0808980106/DCSupplemental.

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3. Hancock JT,
4. Hiscock SJ
(2006) Production of reactive oxygen species and reactive nitrogen species by angiosperm stigmas and pollen: Potential signalling crosstalk? New Phytol 172:221–228.
OpenUrl CrossRef PubMed
↵
1. Potocky M,
2. et al.
(2007) Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth. New Phytol 174:742–751.
OpenUrl CrossRef PubMed
↵
1. Overmyer K,
2. Brosche M,
3. Kangasjärvi J
(2003) Reactive oxygen species and hormonal control of cell death. Trends Plants Sci 8:335–342.
OpenUrl CrossRef
↵
1. Miller G,
2. Mittler R
(2006) Could heat shock transcription factors function as hydrogen peroxide sensors in plants? Ann Bot 98:279–288.
OpenUrl Abstract/FREE Full Text
↵
1. Despres C,
2. et al.
(2003) The Arabidopsis NPR1 disease resistance protein is a novel cofactor that confers redox regulation of DNA binding activity to the basic domain/leucine zipper transcription factor TGA1. Plant Cell 15:2181–2191.
OpenUrl Abstract/FREE Full Text
↵
1. Davletova S,
2. et al.
(2005) Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis. Plant Cell 17:268–281.
OpenUrl Abstract/FREE Full Text
↵
1. Bozhkov PV,
2. Filonova LH,
3. Suarez MF
(2005) Programmed cell death in plant embryogenesis. Curr Top Dev Biol 67:135–179.
OpenUrl CrossRef PubMed
↵
1. Jabs T,
2. Dietrich RA,
3. Dangl JL
(1996) Initiation of runaway cell death in an Arabidopsis mutant by extracellular superoxide. Science 273:1853–1856.
OpenUrl Abstract/FREE Full Text
↵
1. Torres MA,
2. Dangl JL,
3. Jones JD
(2002) Arabidopsis gp91^phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci USA 99:517–522.
OpenUrl Abstract/FREE Full Text
↵
1. Goldman MH,
2. Goldberg RB,
3. Mariani C
(1994) Female sterile tobacco plants are produced by stigma-specific cell ablation. EMBO J 13:2976–2984.
OpenUrl PubMed
↵
1. Verhoeven T,
2. et al.
(2005) STIG1 controls exudate secretion in the pistil of petunia and tobacco. Plant Physiol 138:153–160.
OpenUrl Abstract/FREE Full Text
↵
1. Tang W,
2. Kelley D,
3. Ezcurra I,
4. Cotter R,
5. McCormick S
(2004) LeSTIG1, an extracellular binding partner for the pollen receptor kinases LePRK1 and LePRK2, promotes pollen tube growth in vitro. Plant J 39:343–353.
OpenUrl CrossRef PubMed
↵
1. Overmyer K,
2. et al.
(2008) Complex phenotypic profiles leading to ozone sensitivity in Arabidopsis thaliana mutants. Plant Cell Environ 31:1237–1249.
OpenUrl CrossRef PubMed
↵
1. Wu HM,
2. Cheung AY
(2000) Programmed cell death in plant reproduction. Plant Mol Biol 44:267–281.
OpenUrl CrossRef PubMed
↵
1. Berg RH,
2. Beachy RN
(2008) Fluorescent protein applications in plants. Methods Cell Biol 85:153–177.
OpenUrl CrossRef PubMed
↵
1. Kollist T,
2. et al.
(2007) A novel device detects a rapid ozone-induced transient stomatal closure in intact Arabidopsis and its absence in abi2 mutant. Physiol Plant 129:796–803.
OpenUrl CrossRef
↵
1. Overmyer K,
2. et al.
(2000) Ozone-sensitive Arabidopsis rcd1 mutant reveals opposite roles for ethylene and jasmonate signaling pathways in regulation superoxide-dependent cell death. Plant Cell 12:1849–1862.
OpenUrl Abstract/FREE Full Text
↵
1. Overmyer K,
2. et al.
(2005) Ozone-induced programmed cell death in the Arabidopsis radical-induced cell death1 mutant. Plant Physiol 137:1092–1104.
OpenUrl Abstract/FREE Full Text
↵
1. Schmelz EA,
2. et al.
(2003) Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants. Proc Natl Acad Sci USA 100:10552–10557.
OpenUrl Abstract/FREE Full Text
↵
1. Hellens RP,
2. et al.
(2000) pGreen: A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832.
OpenUrl CrossRef PubMed
↵
1. Clough SJ,
2. Bent AF
(1998) Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743.
OpenUrl CrossRef PubMed
↵
1. Yu IC,
2. Parker J,
3. Bent AF
(1998) Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant. Proc Natl Acad Sci USA 95:7819–7824.
OpenUrl Abstract/FREE Full Text
↵
1. Larkin MA,
2. et al.
(2007) Clustal W and clustal X version 2.0. Bioinformatics 23:2947–2948.
OpenUrl Abstract/FREE Full Text
↵
1. Tamura K,
2. Dudley J,
3. Nei M,
4. Kumar S
(2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599.
OpenUrl Abstract/FREE Full Text

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Proceedings of the National Academy of Sciences: 106 (13)

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Article Classifications

Biological Sciences
Plant Biology

[1] ↵
Kangasjärvi J,
Jaspers P,
Kollist H
(2005) Signalling and cell death in ozone-exposed plants. Plant Cell Environ 28:1021–1036.
OpenUrl CrossRef

[2] Kangasjärvi J,

[3] Jaspers P,

[4] Kollist H

[5] ↵
Valko M,
et al.
(2007) Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39:44–84.
OpenUrl CrossRef PubMed

[6] Valko M,

[7] et al.

[8] ↵
Vahisalu T,
et al.
(2008) SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling. Nature 452:487–491.
OpenUrl CrossRef PubMed

[9] Vahisalu T,

[10] et al.

[11] ↵
Carol RJ,
Dolan L
(2006) The role of reactive oxygen species in cell growth: Lessons from root hairs. J Exp Bot 57:1829–1834.
OpenUrl Abstract/FREE Full Text

[12] Carol RJ,

[13] Dolan L

[14] ↵
McInnis SM,
Desikan R,
Hancock JT,
Hiscock SJ
(2006) Production of reactive oxygen species and reactive nitrogen species by angiosperm stigmas and pollen: Potential signalling crosstalk? New Phytol 172:221–228.
OpenUrl CrossRef PubMed

[15] McInnis SM,

[16] Desikan R,

[17] Hancock JT,

[18] Hiscock SJ

[19] ↵
Potocky M,
et al.
(2007) Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth. New Phytol 174:742–751.
OpenUrl CrossRef PubMed

[20] Potocky M,

[21] et al.

[22] ↵
Overmyer K,
Brosche M,
Kangasjärvi J
(2003) Reactive oxygen species and hormonal control of cell death. Trends Plants Sci 8:335–342.
OpenUrl CrossRef

[23] Overmyer K,

[24] Brosche M,

[25] Kangasjärvi J

[26] ↵
Miller G,
Mittler R
(2006) Could heat shock transcription factors function as hydrogen peroxide sensors in plants? Ann Bot 98:279–288.
OpenUrl Abstract/FREE Full Text

[27] Miller G,

[28] Mittler R

[29] ↵
Despres C,
et al.
(2003) The Arabidopsis NPR1 disease resistance protein is a novel cofactor that confers redox regulation of DNA binding activity to the basic domain/leucine zipper transcription factor TGA1. Plant Cell 15:2181–2191.
OpenUrl Abstract/FREE Full Text

[30] Despres C,

[31] et al.

[32] ↵
Davletova S,
et al.
(2005) Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis. Plant Cell 17:268–281.
OpenUrl Abstract/FREE Full Text

[33] Davletova S,

[34] et al.

[35] ↵
Bozhkov PV,
Filonova LH,
Suarez MF
(2005) Programmed cell death in plant embryogenesis. Curr Top Dev Biol 67:135–179.
OpenUrl CrossRef PubMed

[36] Bozhkov PV,

[37] Filonova LH,

[38] Suarez MF

[39] ↵
Jabs T,
Dietrich RA,
Dangl JL
(1996) Initiation of runaway cell death in an Arabidopsis mutant by extracellular superoxide. Science 273:1853–1856.
OpenUrl Abstract/FREE Full Text

[40] Jabs T,

[41] Dietrich RA,

[42] Dangl JL

[43] ↵
Torres MA,
Dangl JL,
Jones JD
(2002) Arabidopsis gp91^phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci USA 99:517–522.
OpenUrl Abstract/FREE Full Text

[44] Torres MA,

[45] Dangl JL,

[46] Jones JD

[47] ↵
Goldman MH,
Goldberg RB,
Mariani C
(1994) Female sterile tobacco plants are produced by stigma-specific cell ablation. EMBO J 13:2976–2984.
OpenUrl PubMed

[48] Goldman MH,

[49] Goldberg RB,

[50] Mariani C

[51] ↵
Verhoeven T,
et al.
(2005) STIG1 controls exudate secretion in the pistil of petunia and tobacco. Plant Physiol 138:153–160.
OpenUrl Abstract/FREE Full Text

[52] Verhoeven T,

[53] et al.

[54] ↵
Tang W,
Kelley D,
Ezcurra I,
Cotter R,
McCormick S
(2004) LeSTIG1, an extracellular binding partner for the pollen receptor kinases LePRK1 and LePRK2, promotes pollen tube growth in vitro. Plant J 39:343–353.
OpenUrl CrossRef PubMed

[55] Tang W,

[56] Kelley D,

[57] Ezcurra I,

[58] Cotter R,

[59] McCormick S

[60] ↵
Overmyer K,
et al.
(2008) Complex phenotypic profiles leading to ozone sensitivity in Arabidopsis thaliana mutants. Plant Cell Environ 31:1237–1249.
OpenUrl CrossRef PubMed

[61] Overmyer K,

[62] et al.

[63] ↵
Wu HM,
Cheung AY
(2000) Programmed cell death in plant reproduction. Plant Mol Biol 44:267–281.
OpenUrl CrossRef PubMed

[64] Wu HM,

[65] Cheung AY

[66] ↵
Berg RH,
Beachy RN
(2008) Fluorescent protein applications in plants. Methods Cell Biol 85:153–177.
OpenUrl CrossRef PubMed

[67] Berg RH,

[68] Beachy RN

[69] ↵
Kollist T,
et al.
(2007) A novel device detects a rapid ozone-induced transient stomatal closure in intact Arabidopsis and its absence in abi2 mutant. Physiol Plant 129:796–803.
OpenUrl CrossRef

[70] Kollist T,

[71] et al.

[72] ↵
Overmyer K,
et al.
(2000) Ozone-sensitive Arabidopsis rcd1 mutant reveals opposite roles for ethylene and jasmonate signaling pathways in regulation superoxide-dependent cell death. Plant Cell 12:1849–1862.
OpenUrl Abstract/FREE Full Text

[73] Overmyer K,

[74] et al.

[75] ↵
Overmyer K,
et al.
(2005) Ozone-induced programmed cell death in the Arabidopsis radical-induced cell death1 mutant. Plant Physiol 137:1092–1104.
OpenUrl Abstract/FREE Full Text

[76] Overmyer K,

[77] et al.

[78] ↵
Schmelz EA,
et al.
(2003) Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants. Proc Natl Acad Sci USA 100:10552–10557.
OpenUrl Abstract/FREE Full Text

[79] Schmelz EA,

[80] et al.

[81] ↵
Hellens RP,
et al.
(2000) pGreen: A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832.
OpenUrl CrossRef PubMed

[82] Hellens RP,

[83] et al.

[84] ↵
Clough SJ,
Bent AF
(1998) Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743.
OpenUrl CrossRef PubMed

[85] Clough SJ,

[86] Bent AF

[87] ↵
Yu IC,
Parker J,
Bent AF
(1998) Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant. Proc Natl Acad Sci USA 95:7819–7824.
OpenUrl Abstract/FREE Full Text

[88] Yu IC,

[89] Parker J,

[90] Bent AF

[91] ↵
Larkin MA,
et al.
(2007) Clustal W and clustal X version 2.0. Bioinformatics 23:2947–2948.
OpenUrl Abstract/FREE Full Text

[92] Larkin MA,

[93] et al.

[94] ↵
Tamura K,
Dudley J,
Nei M,
Kumar S
(2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599.
OpenUrl Abstract/FREE Full Text

[95] Tamura K,

[96] Dudley J,

[97] Nei M,

[98] Kumar S

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