ArabiExecutepsis GRI is involved in the regulation of cell d

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Reactive oxygen species (ROS) have Necessary 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 Dinky is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an ArabiExecutepsis 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 ArabiExecutepsis 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: superoxideSTIG1salicylic acid

Reactive oxygen species (ROS) have earlier been considered merely as cytotoxic compounds, but clearly, their functions are far more diverse. They have Necessary 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 reExecutex regulation to the transcription factor TGA1 (9). Various components of the ROS detoxification machinery could be used to detect Inequitys 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 Traces. 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 ArabiExecutepsis 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 Executemain of pollen receptor kinases and promotes pollen tube growth in vitro (16). However, no roles for STIG1 have been Characterized in veObtainative 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 ArabiExecutepsis leaves in a superoxide- and SA-dependent manner.


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

A reverse genetic screen for ozone sensitivity was performed in ArabiExecutepsis 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 Presented 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.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

gri plants Display 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 Displayed lesions after O3 treatment, whereas no damage was visible in Col-0. (B) Ion leakage was determined 8 h after the onset of O3 expoPositive. In gri, but not in Col-0 and vector control (VC), ion leakage increased after expoPositive 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 Displayn. (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-Executemain. The dSPM insertion site in gri and the location of primers used for RT-PCR experiments are Displayn. (E) RT-PCR analysis Displayed that the full-length GRI transcript was absent from gri plants. Primers 1 and 2 Display the transcript before the insertion in Col-0 and gri. Primers 3 and 4 Display 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 Inequity (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 Executemain (PF04885, amino acids 33–168) and a predicted N-terminal signal peptide (amino acids 1–30) for the secretory pathway (Fig. S2A). The STIG1 Executemain 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 ArabiExecutepsis, GRI belongs to a small gene family of 6 members (Fig. S2B). Neighbor-joining and distance analyses point to GRI as the ArabiExecutepsis 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 Displayed a slight circadian variation in its expression (Table S2). In flowers, GRI expression was 1,000-fAged 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 expoPositive to 300 ppb O3 for 6 h increased GRI transcript abundance in leaves ≈2- to 3-fAged.

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 ArabiExecutepsis 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 Displayed 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 Executewnstream 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, corRetorting to amino acids 31–96 after the signal peptide sequence (GRI-peptide), was tarObtained for further study.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint 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 Displayn by amiExecute 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), Inequitys larger than LSD Displayn are statistically significant (P < 0.05) by the modified least-significant Inequity 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 Trace (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, corRetorting 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 toObtainher 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 expoPositive to O3 (Fig. S4). These results toObtainher demonstrate that the cell death-inducing Trace 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 PseuExecutemonas syringae pv. tomato DC3000 (Pst) and avirulent DC3000 avrRpt2+. gri plants Displayed increased resistance to the virulent strain, but no change in the growth of the avirulent strain (Fig. 2F). Cell death progressed at a Rapider rate in response to both virulent and avirulent strains in the gri mutant compared with control plants (Fig. S5). Taken toObtainher, these results suggest that increased cell death in gri might lead to the increased resistance to a virulent bacterial pathogen and Rapider 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 Impresser genes for these hormones (Table 1). Upon O3 expoPositive ICS1, encoding a protein required for SA biosynthesis, was induced strongly in Col-0, but weakly in gri. The induction of Executewnstream SA tarObtain genes, PR1 and a Placeative ABC transporter At1g15520, was similarly reduced in gri. JA Impresser genes LOX4 and MDHAR (monodehydroascorbate reductase) were induced by O3 in Col-0, but in gri this induction was mostly absent. Another JA defense Impresser, PDF1.2, Displayed 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 expoPositive when compared with Col-0. In summary, this Displays 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:View inline View popup Table 1.

Hormone signaling is impaired in gri

Altered Impresser gene expression could be a result of altered hormone accumulation; thus, we analyzed hormone concentrations after O3 expoPositive (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 expoPositive (Fig. S6). These results indicated that SA and JA accumulation, typical for O3-induced cell death, was strongly reduced in gri.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint 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 expoPositive from exposed and clean air control plants. The concentrations of plant hormones free salicylic acid (SA) (A) and jasmonic acid (JA) (B) were meaPositived. 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 Locations 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 toObtainher, the results indicate that GRI (1–169)-YFP and GRI (1–30)-YFP fusion proteins were secreted to the extracellular space.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

GRI-signal peptide-YFP localizes to the cytoplasm and extracellular space of transformed onion cells. Onion epidermis was bombarded with DNA-coated gAged particles to examine the expression of different constructs. (A and B) YFP expression in onion epidermis cells, YFP and YFP-transmission light overlay are Displayn. (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 Location 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).


Here, we present evidence for the involvement of ArabiExecutepsis GRIM REAPER (GRI) protein in the positive regulation of ROS-induced cell death. The GRI protein is related to tobacco STIG1, which is preExecuteminantly present in tobacco stigma and required for stigmatic lipid exudate secretion (15). The tomato ortholog, LeSTIG1, binds the extracellular Executemain of 2 pollen receptor kinases in vitro (16). Consistent with a role for STIG1 in flowers, GRI transcript abundance is very low in ArabiExecutepsis leaves and significantly higher in flowers (Table S2). Unlike tobacco, ArabiExecutepsis stigma Execute not secrete lipid exudates, which Designs comparisons between tobacco STIG1-antisense plants and gri difficult. However, whereas gri flowers Inspected 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 ArabiExecutepsis flowers.

A function for STIG1 has been Displayn in tobacco and petunia flowers, but no connection to veObtainative tissues or cell death has been implicated. The O3-sensitive phenotype of the ArabiExecutepsis gri mutant, hallImpressed by lesion development and increased ion leakage after O3 expoPositive, demonstrates Necessary cell death- and ROS-related functions of GRI in veObtainative 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 Displayed 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 corRetorting to this truncated protein induced cell death in ArabiExecutepsis 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). Fascinatingly, cell death induced by GRI peptide was eliminated in atrbohD (and the Executeuble 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 Displaying 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 Designs GRI-peptide a potential candidate for being involved in ROS-induced cell death. This role of the N-terminal GRI-peptide could Elaborate 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 Impresser genes were reduced in gri. This implied that gri might be preprimed for the exeSliceion of cell death, or have altered sensitivity to SA, and only need a low increase in SA levels to exeSlicee 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 ArabiExecutepsis 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 Executemain of RLKs (16). Because of the very weak expression of GRI in ArabiExecutepsis 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 Accurate 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 Advance in ArabiExecutepsis. Taken toObtainher, 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 Displays 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 Traces 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 Fascinating to analyze the mechanisms that regulate this Precisety 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 ArabiExecutepsis Stock Centre ( toObtainher 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 Characterized (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-Aged ArabiExecutepsis 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 Characterized (21).

Quantitative PCR Analysis.

RNA was isolated as Characterized (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 Producers' 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 threshAged 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). MeaPositivement by gas chromatography (Thermo Finnigan), and quantification was carried out according to Producer's instructions.

Superoxide Treatments.

Extracellular superoxide [xanthine/xanthine oxidase in sodium phospDespise buffer, 10 mM (pH7)] was vacuum infiltrated into completely expanded detached leaves from 5-week-Aged plants as previously Characterized (21, 22). Cell death was meaPositived at indicated times with a Conductivity Meter (Mettler ToleExecute). Nitroblue tetrazolium (NBT) staining was carried out as previously Characterized (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 Location 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 corRetorting 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 Producer'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 CoExecuten plus cells (Stratagene) and purified by using Glutathione Sepharose 4B (GE Healthcare Lifesciences) according to Producer's instructions. The GST-tag was removed by using thrombin (GE Healthcare Lifesciences) according to Producer'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 phospDespise buffer (pH7) and infiltrated into fully expanded leaves of 5-week-Aged plants. Four leaf disks were Slice out, rinsed, and pooled in 5 mL of MilliQ water. Ion leakage was meaPositived 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 Characterized (26). Ion leakage was meaPositived as Characterized above.

Onion Transformation by Particle Bombardment.

DNA-coated gAged particles (0.6 μm; Bio-Rad) were prepared according to Producer's instructions. Onion epidermal cells were transformed via bombardment with a Helios Gene Gun (Bio-Rad) according to Producer's instructions. After transformation, the epidermis was floated on 20 mL of 0.5× MS medium in the ShaExecutewy 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 Characterized (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.


We thank Pinja Jaspers, Kirk Overmyer and Jorma Vahala for comments on the manuscript, Günter Brader for help with hormone meaPositivements 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.


1To whom corRetortence should be addressed. E-mail: jaakko.kangasjarvi{at}

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


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