Tomato Phospholipid Hydroperoxide Glutathione Peroxidase Inhibits Cell Death Induced by Bax and Oxidative Stresses in Yeast and Plants1 Shaorong Chen2, Zarir Vaghchhipawala2, Wei Li, Han Asard, and Martin B. Dickman* Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68583 (S.C., Z.V., W.L., M.B.D.); and Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 (H.A.)
Using a conditional life or death screen in yeast, we have isolated a tomato (Lycopersicon esculentum) gene encoding a phospholipid hydroperoxide glutathione peroxidase (LePHGPx). The protein displayed reduced glutathione-dependent phospholipid hydroperoxide peroxidase activity, but differs from counterpart mammalian enzymes that instead contain an active seleno-Cys. LePHGPx functioned as a cytoprotector in yeast (Saccharomyces cerevisiae), preventing Bax, hydrogen peroxide, and heat stress induced cell death, while also delaying yeast senescence. When tobacco (Nicotiana tabacum) leaves were exposed to lethal levels of salt and heat stress, features associated with mammalian apoptosis were observed. Importantly, transient expression of LePHGPx protected tobacco leaves from salt and heat stress and suppressed the apoptoticlike features. As has been reported, conditional expression of Bax was lethal in tobacco, resulting in tissue collapse and membrane permeability to Evans blue. When LePHGPx was coexpressed with Bax, little cell death and no vital staining were observed. Moreover, stable expression of LePHGPx in tobacco conferred protection against the fungal phytopathogen Botrytis cinerea. Taken together, our data indicated that LePHGPx can protect plant tissue from a variety of stresses. Moreover, functional screens in yeast are a viable tool for the identification of plant genes that regulate cell death.
Programmed cell death (PCD) is a genetically controlled process that plays an essential role in the biology of plants and animals (Vaux and Strasser, 1996; Vaux et al., 2001; Dickman and Reed, 2003). Since the early observations of Kerr et al. (1972), which slowly gained acceptance, it has become clear that proper regulation of PCD is crucial for organisms in eliminating cells in a variety of developmental, physiological, and/or pathological contexts. Thus, altruistic cellular suicide is a property of normal physiology and homeostasis, which benefits multicellular organisms. Not only is PCD observed in widely divergent species across broad taxonomic distances, but the molecular components of the death signaling pathways show a high degree of structural and functional conservation, to the extent that cell death regulatory genes from one species (e.g. humans) can function in phylogenetically distant species (e.g. worms; Vaux et al., 1992). In plants, PCD plays a normal physiological role in a number of developmental processes including xylogenesis, senescence, root cap growth, and responses to pathogens (Beers and McDowell, 2001). Though the biochemical mechanisms responsible for cell suicide in plants are largely unknown, a variety of reports 1 This work was supported by the National Science Foundation (grant no. IBN–0133078 to M.B.D.). 2 These authors contributed equally to the paper. * Corresponding author; e-mail
[email protected]; fax 402–472–2853. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.038091.
1630
indicate similarities to the PCD (apoptosis) that occurs in animal species. For example, PCD in plants typically requires new gene expression and thus can be suppressed by cycloheximide and similar inhibitors of protein or RNA synthesis (Havel and Durzan, 1996). The morphological characteristics of plant cells undergoing PCD also bear some striking similarities to apoptosis in animals, though the presence of a cell wall around plant cells imposes certain differences. Akin to animal cells, PCD in plants is associated with internucleosomal DNA fragmentation (DNA ladders) and the activation of proteases (Wang et al., 1996; McCabe et al., 1997; del Pozo and Lam, 1998; Navarre and Wolpert, 1999; Solomon, et al. 1999). Moreover, ectopic expression of certain animal anti-apoptosis genes in transgenic plants has been demonstrated to provide protection from crop pathogens and other insults as a result of cell death suppression (Mitsuhara et al., 1999; Dickman et al., 2001). Conversely, expression of animal pro-apoptotic proteins, such as Bax, in plants can induce a cell death similar to the hypersensitive response program for cell suicide (Lacomme and Santa Cruz, 1999). However, to date, few endogenous plant genes have been identified that share sequence homology with the apoptosis genes of animal cells. With the completion of the Arabidopsis genome, as well as other advancing plant sequencing efforts, it has become clear that core apoptotic pathway members (e.g. Bcl-2 family, caspases) with similarity at the primary sequence level are not present in plant genomes. Thus, alternative approaches are necessary to identify candidate plant genes that regulate apoptotic-like processes. Toward this effort, we have been using baker’s
Plant Physiology, July 2004, Vol. 135, pp. 1630–1641, www.plantphysiol.org 2004 American Society of Plant Biologists
Tomato Phospholipid Peroxidase Inhibits Cell Death
yeast (Saccharomyces cerevisiae) to study regulation of heterologous gene expression with respect to PCD. Yeast has been shown to be a useful model for apoptosis research (Madeo et al., 1999; Jin and Reed, 2002). Reports from our lab and several others have provided evidence that yeast displays several of the hallmark features associated with apoptosis, including chromatin condensation, DNA fragmentation, and externalization of phosphatidylserine (Madeo et al., 1999; Chen et al., 2003). While not all of the defining features of metazoan PCD have been observed in yeast, it has become evident that PCD occurs, exhibiting at least some of these features. Upon examination of the completed genomic sequence of yeast, with one possible exception (Madeo et al., 2002), yeast also has no apparent homologs of major metazoan apoptotic regulators (e.g. Bax/Bcl-2 family, caspases, Apaf-1/CED-4, etc.). Therefore, yeast may be considered a genetically null background system to study interactions between heterologously expressed components of apoptotic pathways (Frohlich and Madeo, 2000). Expression of various apoptotic inducers, including Bax, caspases, p53, or CED-4/ Apaf-1, results in death of yeast (Tao et al., 1999; Jin and Reed, 2002). Coexpression of Bax with Bcl-2 or BclxL inhibits yeast cell death, as observed in animal cells (Zha et al., 1996). In particular, the lethal phenotype observed in Bax expressing yeast has been exploited for structure/function studies, as well as genediscovery efforts by screening for animal genes that suppress Bax-induced lethality (Xu and Reed, 1998). Thus, functional screens using yeast present a potentially useful approach toward the identification of candidate genes that may modulate plant PCD. The yeast Gal/Bax assay is predicated on the ability of ectopically expressed mammalian Bax to kill yeast and on the ability of cytoprotective proteins to rescue yeast from the lethal phenotype conferred by Bax (Jin and Reed, 2002). Here we describe the isolation and functional characterization of a novel tomato (Lycopersicon esculentum) gene identified by the yeast Gal/Bax screen that encodes a phospholipid hydroperoxide glutathione peroxidase (PHGPx). PHGPx have been identified in several plant species, including tomato (Depe`ge et al., 1998; Herbette et al., 2002), and functions in the removal of phospholipid hydroperoxides, which are generated as products of lipoxygenase catalyzed oxygenation of fatty acids (Ursini et al., 1985). Functional studies show that LePHGPx inhibits not only oxidative stress induced cell death in yeast but also inhibits, salt, heat, and Bax induced PCD in tobacco (Nicotiana tabacum) plants.
RESULTS LePHGPx Suppresses Bax Lethality in Yeast
Bax is a pro-apoptotic member of the Bcl-2 family of proteins and has been shown to induce cell death in Plant Physiol. Vol. 135, 2004
mammals, plants, and yeast (Xu and Reed, 1998; Lacomme and Santa Cruz, 1999). To identify plant genes that inhibit Bax-induced lethality in yeast, we screened a tomato cDNA library as described in ‘‘Materials and Methods.’’ Transformants were selected by plating transformed cells on Gal-containing solid medium to induce Bax expression. Viable transformants presumably contain cDNAs that can suppress the Bax cytotoxicity by overexpression of a tomato cDNA. Since a single yeast transformant may contain several types of plasmids, the cDNA that is actually responsible for suppressing Bax toxicity is segregated from other irrelevant cDNAs by ‘‘passingthrough-Escherichia coli’’ (Xu and Reed, 1998). The ability of the cDNA to neutralize Bax cytotoxicity was verified by reintroduction of the cDNA into Gal1Bax-bearing yeast cells. Some of the cDNAs encoded proteins that somehow interfered with the expression of the Gal1 promoter as opposed to blocking the function of Bax, necessitating that each candidate clone be tested for suppression of a Gal1-lacZ gene. Those cDNAs that tested positive for suppression of Bax function but not Gal1 promoter expression were then taken forward to immunoblot analysis, where we verified that they do not interfere with Bax protein production in yeast. From 5 3 106 transformants, hundreds of colonies were obtained that grew on Galcontaining plates, which induces Bax expression and
Figure 1. The tomato LePHGPx gene product inhibits Bax-induced cell death in yeast. A, Yeast strain EGY48 carrying plasmids that encode pGilda-Bax plus Bcl-xl (positive control); pGilda-Bax plus vector pB42AD, or pGilda-Bax plus LePHGPx were spotted on plates at 5-fold dilutions. Expression of Bax was induced by Gal, whereas LePHGPx and Bcl-xl expression was constitutive. B, Yeast growth curves for the strains described in A. pGilda-Bax plus Bcl-xl (¤); pGilda-Bax plus vector pB42AD (:); pGilda-Bax plus LePHGPx (n). 1631
Chen et al.
normally kills the cells. Of these, 114 were picked at random and restreaked onto Gal plates, resulting in 61 colonies that displayed Bax-resistance. Plasmid DNA was recovered from these 61 yeast transformants and retransformed into yeast harboring the pGilda-Bax plasmid. Induction of Bax expression on Gal revealed that 8 of these 61 candidate Bax-suppressors conferred Bax-resistance phenotypes. Of the 8 clones thus identified, 1 encoded a predicted phospholipid hydroperoxide glutathione peroxidase (LePHGPx), which will be described in more detail below. To show conclusively that LePHGPx rescued yeast from Bax-induced lethality, the yeast strain EGY48 containing plasmids pGilda-Bax and pB42ADLePHGPx were grown, pelleted, washed, and resuspended as described in ‘‘Materials and Methods.’’ After 5 d incubation, it was apparent that yeast carrying LePHGPx was able to grow and survive on media that induce lethal Bax expression (Fig. 1A). The growth rate of EGY48 cells coexpressing Bax and LePHGPx and EGY48 expressing only the Bax or Bax and Bcl-xL was measured at regular intervals up to 48 h. As shown in Figure 1B, expression of LePHGPx enabled the Bax expressing cells to proliferate, albeit at a lower rate than the Bcl-xL expressing cells, which is a bona fide Bax inhibitor. In contrast, Bax expressing cells did not show significant growth. The full length LePHGPx cDNA was sequenced. LePHGPx exhibited 79% identity at the amino acid level with the PHGPx from Momordica charantia (Li et al., 2001), 71% identity with the PHGPx from Citrus sinensi (Holland et al., 1993), and 69% identity with GPXle-1 from tomato (Depe`ge et al., 1998). The alignment of amino acid sequences (Fig. 2A) showed that LePHGPx shares high sequence homology with other plant PHGPx proteins, especially in the three domains (G1–G3), which are signature structural motifs of GPx proteins (Jung et al., 2002). These domains contain highly conserved amino acids, including the presumable active site Cys residue at the position occupied by SeCys in mammalian GPx proteins. The conserved Gly residue in domain G1, the Gln residue in domain G2, and a Trp-Asn-Phe motif in domain G3 are all present in the tomato PHGPx sequence. These residues are believed to form the catalytic triad of GPx proteins (Epp et al., 1983). When the phylogenetic relationship between the amino acid sequence of LePHGPx and those of other GPx isoforms is viewed as a
Figure 2. Amino acid alignment and phylogenetic tree of LePHGPx with other GPx polypeptides from plants and humans. A, The deduced amino acid sequences aligned for comparison are from tomato (LePHGPx), tomato (GPXle-1), Momordica chavantia (McGPx), Arabidopsis (AtGPx1 and AGPpx2), spinach (SoGPPx), cotton (GhGPx), Citrus sinensis (CsGpx), Brassicca napus (BnGPx), Pisum sativum 1632
(PsGpx), human (HsGPx), and Mus musculus (MmGPx). Sequences were obtained from the Swiss Prot database. The conserved three Cys residues of plant Bax proteins that correspond to Cys-40, Cys-69, and Cys-88 are indicated by inverted triangles. The SeCys residues of the mammalian PHGPxs HsGPx and MmGPx are denoted by periods, and the highly conserved G1, G2, and G3 regions are boxed. B, A phylogenetic tree was generated from the sequences in A. The sequences were aligned and compared to construct the tree using (1) mammalian GPx, (2) nonspecific targeted GPx, and (3) plastid targeted GPX. Plant Physiol. Vol. 135, 2004
Tomato Phospholipid Peroxidase Inhibits Cell Death
concentration of H2O2 was increased to 9 mM, almost all of the yeast cells (wild type and transformed) were killed. For heat stress assays, yeast harboring LePHGPx were pretreated at 37C for 30 min and then heat shocked at 50C for 30 min. Under these conditions, in wild-type cells the viability was 16%, but the LePHGPx expressing cells had a 52% level of viability (Fig. 3B). LePHGPx Delays Senescence in Yeast
Figure 3. LePHGPx protects yeast from H2O2 and heat stress induced cell death. A, H2O2: Yeast strains containing CED-9 (dark stipples); pB42AD vector alone (hatching) or LePHGPx (light stipples) were treated with 3 mM or 9 mM H2O2 as described in ‘‘Materials and Methods.’’ Surviving colonies were counted and compared to untreated wild type control (not shown). B, Heat stress: Yeast cells containing the same constructs as in A were either preheated (37C) and heat shocked (50C) or just heat shocked. Percent viability was calculated as in A. Data are presented as the average of three experiments with the SE.
dendrogram, it is apparent that LePHGPx belongs to the same branch of the phylogenetic tree as other PHGPx proteins and that the PHGPx group, as a whole, is distinct from other GPx isoforms (Fig. 2B).
Senescence occurs in all organisms, and a number of studies suggest that reactive oxygen species participate in senescence (Lee et al., 1999; Serrano and Blasco, 2001). This process is genetically programmed and thus may be apoptotic-like. The median life span of most laboratory strains of yeast is about 3 d (Jazwinski, 1993). To evaluate the senescence profile of selected yeast strains, cells were grown from lag phase to log phase to stationary phase and the viability of control yeast strains and transformants was compared. The doubling times of all yeast strains were nearly equivalent (4.7 6 0.3 h). Aliquots of cells were removed at specified intervals up to 48 h and the A600 was measured. Senescence was determined by monitoring cell viability after 72 h continuing to 288 h and was measured by plating serial dilutions of the yeast cultures onto YPD plates. The cell density was approximately equivalent between samples at the start of the senescence evaluation. LePHGPx expression significantly delayed the progression of yeast into senescence, relative to the wild-type control and the negative control CED-9 expressing yeast (Fig. 4 and Chen et al., 2003). LePHGPx Displays Reduced Glutathione-Dependent GPx Activity
In order to determine whether the LePHGPx gene encodes a functional enzyme and to evaluate its
LePHGPx Overexpression Protects against H2O2 and Heat Stress in Yeast
LePHGPx-expressing yeast cells were treated with 3 mM or 9 mM H2O2 for 6 h and washed extensively prior to subsequent transfer into yeast peptone dextrose (YPD) plates. During the 6 h treatment, H2O2 concentrations in the medium were still greater than 50% from the starting levels of wild type and LePHGPx expressing yeast cells as determined by the Guiacol assay (Tiedmann, 1997; data not shown). The number of growing colonies was counted after 48 h of incubation at 30C, and the percentage of surviving cells was calculated. Expression of the LePHGPx significantly protected EGY48 cells from death induced by 3 mM H2O2 (Fig. 3A). Of the cells with LePHGPx, 38% remained viable compared to only 8% of the yeast cells containing the vector alone. CED-9 containing yeast served as a positive control (Chen et al., 2003). As the Plant Physiol. Vol. 135, 2004
Figure 4. LePHGPx delays senescence in yeast. Yeast strains as described in Figure 3 were grown to stationary phase, and viability was determined by colony counts of the control yeast strain (vector alone, :) compared to transformants containing CED-9 (¤) or LePHGPx (n). Data are presented as the average of three experiments with the SE. 1633
Chen et al.
LePHGPx Transient Expression Prevents Bax-Induced Cell Death in Tobacco
Table I. Specific activities of recombinant LePHGPX toward different substrates Enzymatic activities were determined in affinity purified proteins after removal of the GST tag. Results of two independent experiments are shown, with their SE and the number of replicate measurements. Substrates are H2O2 (200 mM), Cu-OOH (200 mM), tBu-OOH (200 mM), and PL-OOH (800 mM). Substrates
Specific Activity Sample I
Sample II nmol min
H2O2 Cu-OOH tBu-OOH PL-OOH
6 110 57 2.4
6 6 6 6
2(2) 5(3) 18(3) 0.3(2)
21
mg
21
8 144 34 6
6 6 6 6
5(4) 28(3) 1(2) 2(4)
substrate specificity, recombinant enzymes were prepared in E. coli BL21 cells. Purified LePHGPx showed a single band of about 19 kD in denaturing SDS-PAGE gels (data not shown). LePHGPx displayed GPx activities and catalyzed reduced glutathione-dependent reduction of H2O2, cumene hydroperoxide, t-butyl hydroperoxide (200 mM each), and phospholipid hydroperoxide (PL-OOH; approximately 800 mM; Table I). Specific activities with PL-OOH were low when compared to other substrates but in the same order of magnitude as PHGPx activities from citrus, tobacco, and sunflower (Beeor-Tzahar et al., 1995; Herbette et al., 2002). No activity was observed with extracts from E. coli transformed with the empty vector and affinity purified on glutathione S-transferase (GST)-coupled agarose.
We next determined whether LePHGPx could protect plant cells from Bax-induced cell death as it does in yeast. Bax expression was conditionally regulated by the dexamethasone-inducible promoter. Bax infiltrated tissue showed clear indications of cell death as indicated by tissue collapse, loss of chlorophyll, and membrane permeability to the vital stain, Evans blue (Fig. 6A). Similarly treated tobacco coexpressing LePHGPx 1 Bax showed either no cell death or very small sectors of cell death in the infiltrated areas as indicated by Evans blue staining (compare Fig. 6, A and B). Control plant tissue expressing vector only or mock infiltrations did not show any indications of cell death, nor did such tissue stain with Evans blue,
Endogenous LePHGPx RNA Expression in Tomato Following Stress
Tomato plants were exposed to abiotic and biotic stresses, and RNA was extracted at selected time points and hybridized to radioactively labeled LePHGPx. Since PHGPx is part of a gene family in tomato (Depe`ge et al., 1998) and Arabidopis (Rodriguez Milla et al., 2003) with relatively high levels of sequence similarity (Fig. 2A), LePHGPx specific probes were designed from 3# untranslated regions of the gene, as decribed in ‘‘Materials and Methods.’’ In all treatments, control leaves showed low, basal levels of LePHGPx expression. However, upon exposure to heat, cold, or salt, a considerable increase in transcript levels was observed (Fig. 5, A–C). In a similar manner, abscisic acid (ABA) treatment also induced expression of LePHGPx (Fig. 5D). When tomato leaves were inoculated with two necrotrophic compatible fungal pathogens, Sclerotinia sclerotiorum and Alternaria alternata, again, the induction of LePHGPx expression was evident (Fig. 5E). It should be noted that when leaves were wounded, an increase in this transcript also occurred (not shown). Taken together, biotic and abiotic stresses induce transcriptional activity of LePHGPx. 1634
Figure 5. RNA expression analysis of LePHPx in wild-type tomato plants following various stress treatments. A, LePHGPx expression in tomato after salt treatment. Lane 1, RNA from control plants treated with water; Lanes 2 and 3, RNA from plants 24 and 48 h after 300 mM NaCl application. B, LePHGPx expression after heat exposure of tomato plants to 55C for 20 min. Lane 1, RNA from untreated control plants; Lanes 2, 3, and 4, RNA from plants at 0, 6, and 12 h after heat shock. C, LePHGPx expression after exposure of tomato plants to cold stress (4C for 3 h). Lane 1, RNA from untreated control plants; Lanes 2, 3, and 4, RNA plants at 0, 6, and 12 h after cold treatment. D, LePHGPx expression after ABA treatment. Lane 1, RNA from control plants treated with 0.1% ethanol; Lanes 2, 3, and 4, RNA from plants at 12, 24, and 48 h after 250 mM ABA application respectively. E, LePHGPx expression in tomato after fungal after inoculation. Lane 1, RNA was extracted from control plants inoculated with agar plugs; Lanes 2 and 3, RNA from leaves 4 and 16 h after S. sclerotiorum inoculation; Lanes 4 and 5, RNA from leaves at 24 and 48 h after A. alternata inoculation. Gels were run and blots were hybridized as described in ‘‘Materials and Methods.’’ Plant Physiol. Vol. 135, 2004
Tomato Phospholipid Peroxidase Inhibits Cell Death
Figure 6. Transient expression of LePHGPx rescues BAX induced cell death in tobacco leaves. A, Induction of BAX expression leads to visible loss of chlorophyll, tissue collapse, and cell death. Evans blue staining of tissue showing loss of membrane integrity. B, Leaf sectors coinfiltrated with BAX1LePHGPx (1:1, A600 5 0.6) show minimal or no cell death after 36 h. Evans blue vital staining is generally excluded.
indicating that the cell membranes were intact. These results show that LePHGPx functions to protect tobacco cells from the cell death pathway induced by Bax.
LePHGPx Transient Expression Protects Plant Cells against Abiotic Stress
Since it has been shown that LePHGPx protein levels increase during salt stress (Beeor-Tzahar et al., 1995; Gueta-Dahan et al., 1997), we were interested in determining whether this increase in expression was possibly related to an increased resistance to oxidative stress conditions as observed with yeast. Leaf discs transiently expressing LePHGPx were exposed to 350 mM salt concentrations. After 48 h treatment, empty vector-infiltrated tobacco leaf discs showed a significant yellowing indicative of loss of chlorophyll, while the LePHGPx expressing leaf tissue appeared green with only slight losses of chlorophyll (Fig. 7A and data not shown). DNA extracted from 3 independent transient assays, after 48 h salt treatment, showed DNA laddering in the vector and mock infiltrated samples, while the DNA from LePHGPx expressing leaves was intact (Fig. 7B). In addition, wild type, but not LePHGPx expressing cells, stained positively for the transferase-mediated dUTP nick end labeling (TUNEL) reaction (Fig. 7C), indicating DNA fragmentation had occurred with morphologies reminiscent of animal apoptotic bodies (Fig. 7C, arrowhead). These results indicate that tobacco leaves exposed to lethal doses of salt exhibit features that resemble apoptotic-like cell death and that expression of Plant Physiol. Vol. 135, 2004
LePHGPx confers protection against salt injury and prevents DNA fragmentation. Excessive heat exposure is also known to cause oxidative stress (Samali et al., 1999). We therefore were interested in determining whether LePHGPx expression could protect plant tissue from lethal exposure to heat. Empty vector infiltrated leaf samples subjected to heat stress showed a progression from yellowing of leaves to browning with leaf tissue showing dead cells over more than one-third of the leaf area. Comparatively, LePHGPx infiltrated leaves showed minimal heat damage as indicated by browning being restricted to the leaf margins (Fig. 8). LePHGPx Stable Expression Protects Plant Cells against Biotic Stress
Transgenic tobacco plants were generated harboring single copy number, kanamycin-resistant LePHGPx. A minimum of 5 leaves from 3 independent transformed lines of 5-week-old transgenic tobacco harboring LePHGPx or empty vector were inoculated with the broad host range necrotrophic fungus, Botrytis cinerea. Five-millimeter-diameter agar plugs containing actively growing hyphal tips from 3-d-old colonies of B. cinerea were placed on detached leaves. When transgenic tobacco harboring LePHGPx was inoculated with B. cinerea, leaves were highly resistant to infection and showed only tiny necrotic spots around the agar plugs (Fig. 9). In contrast, leaves transformed with vector alone were highly susceptible and the damaged cells extended far from the agar plugs (Fig. 9). Similar results were obtained with S. sclerotiorum, also a broad, host range necrotroph (data not shown). 1635
Chen et al.
Figure 7. Transient expression of LePHGPx provides protection against salt-induced PCD in tobacco leaf discs. A, Phenotype of leaf discs transiently expressing LePHGPx (left section) and empty vector (right section) after exposure to 350 mM salt for 48 h. Note loss of chlorophyll in empty vector infiltrated leaf discs. B, DNA ladder formation during salt induced apoptosis in control samples. Mock and vector infiltrated samples show DNA fragmentation after 48 h exposure to salt stress while LePHGPx expressing tobacco DNA is intact. C, Propidium iodide and TUNEL staining of leaf discs from empty vector (top sections) and LePHGPx expressing tobacco (bottom sections). Leaf samples were exposed to salt stress for 48 h. Note formation of apoptotic-like bodies (inset, arrowhead) in vectorinfiltrated samples.
Thus, expression of LePHGPx confers protection from both abiotic and biotic stresses.
DISCUSSION
Under aerobic conditions, cells are constantly exposed to the possibility of oxidative damage mediated by reactive oxygen species. Cells possess a range of nonenzymatic and enzymatic defense systems to counter oxidative stress including glutathione, thioredoxin, ascorbate, superoxide dismutase, and peroxidases such as catalases, glutathione peroxidases, and ascorbate peroxidases (Herbette et al., 2002; Moon et al., 2002). In mammals there are at least five isoforms of glutathione peroxidases (GPx), including a phospholipid hydroperoxide GPx (PHGPx or GPx4). PHGPx is a monomeric enzyme, associated with both soluble and membrane fractions, that reduces lipid hydroperoxides (Jung et al., 2002). Phospholipid hydroperoxides are key intermediates in the lipid peroxidation chain reaction, one of the major types of 1636
oxidative damage in cells, associated with membrane perturbation, inactivation of membrane proteins, and cell lysis. Lipid peroxidation has also been linked to pathological conditions such as ischemic injury, apoptosis, atherosclerosis, and carcinogenesis (Nomura et al., 2000). Thus, PHGPx is an important cellular enzyme capable of halting membrane lipid peroxidation and oxidative damage in animal cells. In this report we describe the identification and characterization of a tomato LePHGPx, using a conditional Gal/Bax screen in yeast. Expression of the proapoptotic mammalian Bax gene arrests growth and eventually causes cell death in yeast. This cell death appears to be physiologically relevant since Bax expression induces cytochrome c release from yeast mitochondria and the death phenotype can be rescued by Bcl-2 or Bcl-xl. We and others (Xu and Reed, 1998; Kawai et al., 1999; Chen et al., 2003) have exploited the experimental advantages of yeast as a heterologous system for screening and identification of candidate genes that functionally regulate apoptosis. While plant genes have been identified that inhibit Bax induced Plant Physiol. Vol. 135, 2004
Tomato Phospholipid Peroxidase Inhibits Cell Death
Figure 8. LePHGPx expression protects against heat stress in tobacco leaves. Five days after infiltration, leaves expressing LePHGPx or empty vector were removed from the plant and subjected to heat stress at 55C for 20 min. Leaves were photographed 48 h after heat stress. Empty vector infiltrated leaves show significantly more heat stress induced cell death than LePHGPx expressing leaves.
lethality in yeast, this report extends this observation to plants, thus indicating that function in yeast can translate to function in plants (also see Kawai-Yamada et al., 2001). Importantly, data is presented that indicates that the LePHGPx functions as a cytoprotective protein under various conditions of lethal stress, suggesting that LePHGPx may serve in an analogous manner to animal antiapoptotic genes. Although these results are all based on overexpression of LePHGPx, the northern blots show that the LePHGPx transcript is also up-regulated under diverse lethal stress conditions, suggesting that endogenous LePHGPx is involved in oxidative stress responses. Moreover, heat, cold, salt, and ABA have been shown to induce expression of the citrus PHGPx (Avsian-Kretchmer et al., 1999). A number of genes are similarly induced by these (and other) abiotic stresses, although whether there is a direct relationship for ABA regulating these processes is not clear, as there is evidence for both ABA-dependent and -independent modulation of stress response genes. The induction of LePHGPx by Sclerotinia and Alternaria is of interest, but in the experiments described above, disease ensued. To address whether regulated expression of LePHGPx can confer disease tolerance and/or resistance, transgenic tobacco plants constitutively expressing LePHGPx, were generated. Results clearly indicated that transgenic expression was sufficient to provide cell survival when challenged by the necrotrophic fungus, B. cinerea. This fungus is also known to generate oxidative stress during infection (Kuzniak and Sklodowska, 2004). Our interest in LePHGPx stems from the fact that not only does it inhibit Bax-induced cell death in yeast and that in animals it is involved in ameliorating oxidative stress, but also because PHGPx functions as an anti-apoptotic agent in animals. Moreover, Baxinduced cell death in yeast occurs at least in part by generation of toxic levels of reactive oxygen species (Chen et al., 2003). Involvement of PHGPx in mamPlant Physiol. Vol. 135, 2004
malian signal transduction pathways is suggested by studies of the mitochondrial PHGPx, which functions as an anti-apoptotic agent in mitochondrial death signaling (Nomura et al., 1999, 2000). In plants the role(s) of PHGPx is not well defined. Expression of a PHGPx activity has been shown to increase during exposure to NaCl in citrus (Beeor-Tzahar et al., 1995; Gueta-Dahan, et al., 1997), although the functional implications for these observations are presently unknown. A Chinese cabbage cDNA with sequence similarity to PHGPx has been biochemically characterized and shown to have thioredoxin-dependent peroxidase activity and was suggested to be chloroplast encoded (Jung et al., 2002). The first functional description of a plant protein with PHGPx activity occurred when a tobacco GST/PHGPx was overexpressed. Transgenic seedlings exhibited enhanced growth rates over wild type when exposed to chilling or salt stress (Roxas et al., 1997, 2000). In addition, two PHGPx-like proteins distinct from LePHGPx have been described from tomato and sunflower and were shown to have bifunctional enzyme activities: PHGPxand thioredoxin peroxidase activities (Depe`ge et al., 1998; Herbette et al., 2002). Again, the functional role(s) for these plant enzymes is not clear. It should be mentioned that Predotar (http://Genoplante-info. infobiogen.fr/predotar/ predotar.html) and ChloroP 1.1 (Emanuelsson et al., 1999) ruled out the presence of a predicted chloroplast transit peptide indicating a cytoplasmic subcellular location for LePHGPx. A distinct difference between plant and animal GPx family members is the presence of an active site Cys in plants as compared to animal proteins that contain selenocysteine (SeCys; Stadtman, 1996). The
Figure 9. LePHGPx expression confers protection to the fungal pathogen pathogen B. cinerea. Transgenic tobacco leaves harboring LePHGPx or empty vector were inoculated by placing 5-mm-diameter agar plugs containing actively growing hyphal tips from 3-d-old colonies of B. cinerea grown on potato dextrose agar. Leaves were photographed 4 d after inoculation. Leaves transformed vector alone are significantly more susceptible than LePHGPx expressing leaves. All experiments were repeated three times with similar results. 1637
Chen et al.
importance of the SeCys in the PHGPx catalytic activity has been demonstrated for the animal proteins (Gladyshev et al., 1996). Replacement of the active site Cys by SeCys in a PHGPx from citrus resulted in an increased lipid peroxidase activity, supporting the catalytic role of this residue also in plant PHGPxs (Hazebrouck et al., 2000). The low in vitro activity observed with the recombinant LePHGPx in this study is comparable to that observed with other plant PHGPxs (Beeor-Tzahar et al., 1995; Herbette et al., 2002). The nature of the catalytic activity of the tomato PHGPx is also supported by the high conservation of the GPx motifs in the primary sequence (Fig. 2A). However, the peroxidase activity with PL-OOH as a substrate, and reduced glutathione as the electron donor, is orders of magnitude lower than that of the animal proteins. It is possible that the low level of PL-OOH peroxidase activity of LePHGPx is sufficient to support the protective effect from oxidative lipid damage. LePHGPx was identified by its ability to protect yeast cells during forced expression of Bax. Expression of the pro-apoptotic Bax protein is known to cause depletion of glutathione levels in yeast (Kampranis et al., 2000). This observation has also been noted in response to a number of apoptotic inducers in mammalian systems including infectious disease, FAS, and TNF a (Cai and Jones, 1998). Thus the ability of LePHGPx to protect yeast in the Gal/Bax screen prompted a detailed examination to determine whether or not this enzyme can function as a cytoprotectant in yeast under more physiological stresses and, more importantly, whether these observations could be extended to plants. When yeast was treated with 3 mM H2O2, a concentration which kills yeast in an apoptoticlike manner (Chen et al., 2003), LePHGPx expression considerably prevented cell death from occurring (Fig. 3A). Moreover, TUNEL positively reacting wild-type yeast cells were observed following H2O2 administration, which were absent in the LePHGPx expressing protected yeast strains (data not shown). We also observed a similar situation during heat stress. A common feature of Bax, H2O2, and heat treatments is the generation oxidative stress. Interestingly, LePHGPx also delayed senescence of aging yeast cells grown for extended incubation periods. A crucial question in using heterologous functional screens, such as the one described, is whether candidate plant genes identified in yeast have any meaningful or analogous role in plants. In other words, is the use of yeast screens valid? Significantly, we show a strong correlation in plants to what was observed in yeast, when LePHGPx is expressed under conditions of oxidative stress. We used the pSfinx vector system (Takken et al., 2000) for transient expression of LePHGPx, which combines the advantages of Agrobacterium binary vectors and plant viral vectors. A potato (Solanum tuberosum) virus X (PVX) component with LePHGPx was agro-infiltrated into leaves. The PVX component ensures abundant expression of the gene of interest as well as movement between cells. 1638
Since increases in PHGPx expression has been associated with high salt concentrations, we wanted to see if LePHPGx expression would protect plant tissue from salt stress. Indeed this was shown to be the case, thereby establishing that LePHGPx functions as a cell protectant against salt. Moreover, salt treatment of wild type or mock inoculated plant tissue kills tissue with features associated with mammalian PCD, namely, fragmented DNA resulting in a characteristic ladder. LePHGPx expression also prevented the fragmentation of DNA and maintained membrane integrity. Similar observations occurred during Bax transient expression and coexpression of Bax and LePHGPx. Taken together these data show that yeast screens are a viable tool for the identification of plant genes that regulate cell death. In addition, stable expression of LePHGPx in tobacco plants conferred protection against necrotrophic fungi. Thus, LePHGPx overexpression can protect plants against biotic and abiotic stresses and thus shares properties associated with mammalian anti-apoptotic genes.
MATERIALS AND METHODS Yeast Gal/BAX Assay pGilda-Bax (from John Reed, Burnham Institute, La Jolla, CA; Zha et al., 1996) was transformed into yeast strain EGY-48, and the transformed cells were plated on SD/glu/-his media. Growing colonies were further evaluated for their ability to kill cells on SD/gal/raff/-his media. A tomato cDNA library, constructed from tobacco mosaic virus infected tomato VF36 leaves, was cloned into yeast expression vector pB42AD and transformed into Bax containing yeast cells. The transformed cells were plated on SD/glu/-his/-trp media. Growing cells were collected, washed, and plated on SD/gal/raff/his/-trp media. After 5 d incubation at 30C, the colonies rescued from Bax lethality were streaked onto SD/gal/raff/-his/-trp plates again to confirm the growth phenotype. Tomato cDNAs that conferred resistance to Bax-induced death were plasmid-rescued and the resulting plasmid DNA was retransformed into EGY48 yeast containing pGilda-Bax, and the cells were grown on SD/gal/raff/-his/-trp plates to confirm ability to rescue from Bax-induced lethality. To evaluate yeast cell viability, EGY48 cells containing the plasmids pGilda-Bax and pB42AD, pGilda-Bax and pB42AD-LePHGPx, pGilda-Bax and pB42AD-Bcl-xL, were grown in SD/glu/-his/-trp overnight. The cells were pelleted, washed, and resuspended in water. For the plate assay, the yeast cultures were serial 5-fold diluted, and 5 mL of each dilution was dropped on SD/glu/-his/-Trp or SD/gal/raff/-his/-Trp plates and incubated at 30C for 5 d and photographed. For liquid assays, the cells were resuspended to A600 5 0.05 in SD medium containing 2% Gal and 1% raffinose (gal-raff) as the carbon source, instead of Glc, to induce expression of the fusion proteins from the GAL1 promoter. Aliquots of cells were removed at regular intervals up to 48 h and the A600 was measured.
Yeast Stress Treatment Assays Early log phase yeast cultures (A600 5 0.5) were diluted to a density of A600 5 0.05 with SD/gal-raff/-his or SD/gal-raff/-his/-trp and treated in one of the following ways. For chemical treatment, H2O2 was added at different concentrations and incubated at 30C with vigorous shaking for 6 h. For heat stress, yeast cells were incubated at 37C for 30 min with vigorous shaking then transferred to a water bath at 50C for 5 to 30 min and incubated at 30C with vigorous shaking for 6 h. Following these treatments, viability was determined by plate counting of colony forming units. Ten microliters of cells were sampled, diluted, and spread onto YPD medium with 2% agar then incubated at 30C for 48 h. The number of colonies forming units from treated cells was compared to the colonies forming units of untreated cells. All experiments were repeated at least in triplicate.
Plant Physiol. Vol. 135, 2004
Tomato Phospholipid Peroxidase Inhibits Cell Death
Yeast Growth and Senescence Assay Doubling times were determined by taking early log phase yeast cultures (A600 5 0.5) in SD media containing 2% Gal, 1% raffinose then diluting and incubating at A600 of 0.05 with shaking in the same medium. Aliquots of cells were removed at specified intervals up to 48 h, and the A600 was measured. Senescence was determined by monitoring cell viability after 72 h continuing to 288 h and was measured by plating serial dilutions of the yeast cultures onto YPD plates. Colonies forming units were counted after 2 d incubation at 30C. The cell density was approximately equivalent between samples at the start of the senescence evaluation.
per sample. Plants were kept overnight in the dark after which they were returned to light. Five days after infiltration, 1-cm-diameter discs were excised from infiltrated leaves using a cork-borer and floated on 350 mM NaCl solution, followed by incubation at room temperature under constant illumination. After 48 h, discs were photographed, evaluated for chlorophyll content, and collected for DNA extraction. For heat stress, infiltrated leaves were excised from plant and placed in petri dishes containing moist filter paper. Heat stress was administered at 55C for 20 min, after which the leaf samples were returned to room temperature and placed under constant illumination and high humidity. Leaves were photographed 48 h after application of stress.
Plant Transient Expression Assays
Plant Bax and Fungal Assays
The pSfinx vector system (Takken et al., 2000) was used for plant transient assays. The LePHGPx open reading frame was cloned into the ClaI-AscI sites of the PVX-based vector pSfinx (obtained from Dr. Mathew Joosten, Wageningen University, The Netherlands). Expression was driven by the duplicated PVX coat protein promoter. This construct was electroporated into Agrobacterium tumefaciens MOG101 electrocompetent cells containing the helper plasmid pIC-SArep (Jones et al., 1992) and transformed cells were selected on kanamycin (100 mg/L) and tetracycline (5 mg/L) containing media. A single colony of A. tumefaciens MOG101 harboring the LePHGPx construct was inoculated into 5 mL YEP medium with antibiotics and grown overnight at 30C. Cultures were pelleted and resuspended in induction medium (K2HPO4, 10.5 g/L; KH2PO4, 4.5 g/L; (NH4)2SO4, 1 g/L; sodium citrate, 2H2O, 0.5 g/L; MgSO4, 1 mM; Glc, 0.2%; Glycerol, 0.5%; MES, 10 mM). Prior to use, 50 mg/mL acetosyringone in dimethyl formamide was added to the induction medium. Bacteria were grown in induction medium for 6 to 8 h at 30C, after which cells were pelleted and resuspended in infiltration medium (0.5 3 Murashige and Skoog basal salts/L; 10 mM MES, pH 5.6) at an A600 of 0.8. Acetosyringone (150 mg/mL in dimethyl formamide) was added to infiltration medium just prior to use. Nicotiana tabacum (cv Glurk) plants at the 6-leaf stage were watered prior to infiltration and kept in a growth chamber at 25C. Infiltration was carried out on the underside of healthy young leaves using a needleless tuberculin syringe. Control cultures containing vector only were also infiltrated and a mock infiltration using just infiltration medium was also carried out. Plants were covered and kept in a dark chamber with high humidity. The following day, plants were returned to the growth chamber and kept at 25C for 5 d.
The open reading frames of mouse Bax and LePHGPx were cloned into the XhoI, SpeI sites of the dexamethasone inducible vector pTA7002 (McNellis et al., 1998). These constructs were introduced into the A. tumefaciens strain C58C1 via electroporation, and transformants were selected on media containing kanamycin (50 mg/mL), rifampicin (50 mg/mL), and gentamicin (50 mg/mL). Cultures were prepared as described for the plant transient assays. After 6 to 8 h of growth in induction medium, cells were pelleted and resuspended in infiltration medium (0.5 3 MS-B, 10 mM MES, pH 5.6; add 150 mg/mL acetosyringone prior to use). A600 of cultures was adjusted to 0.6 and tobacco (4–6 leaf stage) leaf sectors were infiltrated with cultures harboring either Bax, LePHGPx, Bax1LePHGPx (1:1), or pTA7002. Mock infiltrations were carried out using infiltration buffer only. After infiltration, plants were kept in the dark overnight, and 24 h later infiltrated sectors were sprayed with 20 mM dexamethasone (with 0.005% Silwet L-77). Leaf tissue was examined at 36 h postinduction, stained with Evans blue (10 mg/mL in PBS) overnight, and cleared in 70% alcohol. To generate transgenic tobacco plants, separate binary vectors were constructed that contained LePHGPx under the control of the cauliflower mosaic virus 35S promoter and the Agrobacterium nopaline synthase terminator. Plant transformation and transgenic evaluations were done as described (Dickman et al., 2001). From three independent transformation events, a minimum of 5 leaves from 5-week-old transgenic tobacco harboring LePHGPx or empty vector were inoculated by placing 5-mm-diameter agar plugs containing actively growing hyphal tips from 3-d-old colonies of Botrytis cinerea grown on potato (Solanum tuberosum) dextrose agar. All experiments were repeated at least three times.
Cell Death Assays To evaluate DNA fragmentation (laddering), leaf tissues, following salt stress, were frozen and ground in liquid nitrogen. DNA was extracted using standard protocols (White and Kaper, 1989). Fifteen micrograms of DNA was loaded onto a 2% agarose gel and electrophoresed at 3 v/cm overnight. Gels were photographed and processed for southern blotting according to standard protocols (Sambrook et al., 1989). Membranes were probed with 32 P-dCTP labeled tobacco genomic DNA. To evaluate DNA fragmentation via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining, plant tissue were fixed with 3.7% fresh formaldehyde, digested with lyticase (5 units/L for 30 min at 37C), then electrostatically bound to a glass slide (Fisherbrand Superfrost Plus, Chicago). Plant tissues were also stained with propidium iodide, a fluorescent DNA stain, which shows that TUNEL labeling occurs specifically with fragmented DNA. The slides were rinsed with phosphate-buffered saline (PBS), incubated in permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) for 2 min on ice, rinsed twice with PBS, incubated with 10 mL TUNEL reaction mixture (200 units/mL terminal deoxynucleotidyl transferase, 1 mM FITC-labeled dUTP, 25 mM Tris/HCl, 200 mM sodium cacodylate, 5 mM cobalt chloride; In Situ cell death detection kit, Fluorescein, Roche, Indianapolis) for 60 min at 37C, and rinsed 3 times with PBS. The slides were then incubated with 1 mg/mL diaminophenylindole in PBS for 10 min at room temperature, rinsed twice with PBS, and analyzed under a fluorescence microscope (Zeiss Axioskop, Jena, Germany) coupled to an imaging system (AxioCam HR, Thornwood, NY).
Salt and/or Heat-Stress Assay Cultures containing the pSfinx vector alone or pSf-LePHGPx were prepared as described above. Infiltration of bacteria was done at A600 5 0.6. Agroinfiltration was carried out on the underside of tobacco (cv Glurk) leaves using a 1-mL tuberculin syringe and up to 4 to 5 leaves were completely infiltrated
Plant Physiol. Vol. 135, 2004
Northern Analysis Three-week-old wild-type Rutger tomato plants grown at 25C with 16 h light periods in greenhouse were used for all RNA expression experiments. For ABA treatments, tomato plants were sprayed with 250 mM of cis,transABA (Sigma, St. Louis) until run off. The ABA solution was prepared from a 1 mM stock solution containing 1% (v/v) ethanol. Control plants were treated with the same solution minus ABA. For salt stress, plants were placed in a solution containing 300 mM NaCl for 24 to 48 h after which RNA was extracted. Control plants were treated similarly with water alone. Plants were exposed to 55C for 20 min for heat stress or 4C for 3 h for cold treatment; plants were then returned to room temperature. RNA was extracted from samples collected before the stress was imposed (control), time 0 (immediately after stress), and 6 and 12 h following stress treatment. To evaluate LePHGPx expression following pathogen challenge, Sclerotinia sclerotiorum and Alternaria alternata were inoculated on the tomato leaves by placing 5-mm-diameter agar plugs containing actively growing hyphal tips from 3- and 7-d-old colonies, respectively. Inoculated plants were placed in a growth chamber at 25C with 16 h light periods and 100% relative humidity. Leaves treated with agar plugs only were used as a control. RNA was extracted as described by Reuber and Ausubel (1996) and northern analysis was done as described by Sambrook et al. (1989). To generate an LePHGPX gene-specific probe, a unique fragment of 200 bp was identified by sequence alignmnet of plant PHGPx genes. This fragment corresponds to the 3# untranslated region and was obtained by PCR using primers: 5#-ttgagcactacaggtgtgaa-3# and 5#-ttactatgcaactttattac-3#.
Enzyme Assays LePHGPx cDNA was subcloned into the EcoRI and XhoI sites of the GST fusion protein expression vector pGEX-4T-1 (Pharmacia Biotech, Piscataway,
1639
Chen et al.
NJ). Expression of GST fusion proteins was carried out as described (Choi et al., 2000). The LePHGPx protein was cleaved by thrombin and purified according to the manufacturer’s manual. LePHGPx activity was determined by using PL-OOH, t-butyl hydroperoxide, cumene hydroperoxide, and H2O2 as the substrates. Enzyme activity was determined as described by Maiorino et al. (1990) by detecting the oxidation of glutathione in a coupled assay using glutathione reductase (Sigma) and saturating concentrations of NADPH. The oxidation of NADPH was recorded at 340 nm in an SLM-Amino DW2000 spectrophotometer in split beam mode at 37C. The activity was calculated using an extinction coefficient of 6,220 M21cm21. Nonspecific NADPH oxidation activity was recorded in the absence of substrates and taken into account in the activity calculation. PL-OOH was synthesized according to Maiorino et al. (1990) using soybean phosphatidyl choline (Avanti PolarLipids, Alabaster, AL) and soybean lipoxygenase (Sigma; Type IV). PL-OOH concentrations were determined as described by El-Saadani et al. (1989). The GenBank accession number for LePHGPx is AY301280. Sequence data from this article have been deposited with the EMBL/ GenBank data libraries under accession number AY301280.
ACKNOWLEDGMENTS We thank Young-ki Park for technical assistance. Received December 23, 2003; returned for revision March 30, 2004; accepted March 30, 2004.
LITERATURE CITED Avsian-Kretchmer O, Eshdat Y, Gueta-Dahan Y, Ben-Hayyim G (1999) Regulation of stress-induced phospholipid hydroperoxide glutathione peroxidase expression in citrus. Planta 209: 469–477 Beeor-Tzahar T, Ben-Hayyim G, Holland D, Faltin Z, Eshdat Y (1995) A stress-associated citrus protein is a distinct plant phospholipid hydroperoxide glutathione peroxidase. FEBS Lett 366: 151–155 Beers EP, McDowell JM (2001) Regulation and execution of programmed cell death in response to pathogens, stress and developmental cues. Curr Opin Plant Biol 4: 561–567 Cai J, Jones DP (1998) Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss. J Biol Chem 273: 11401–11404 Chen SR, Dunigan DD, Dickman MB (2003) Bcl-2 family members inhibit oxidative stress-induced programmed cell death in Saccharomyces cerevisiae. Free Radic Biol Med 34: 1315–1325 Choi IR, Stenger DC, French R (2000) Multiple interactions among proteins encoded by the mite-transmitted wheat streak mosaic tritimovirus. Virology 267: 185–198 del Pozo O, Lam E (1998) Caspases and programmed cell death in the hypersensitive response of plants to pathogens. Curr. Biol. 8: R896 Depe`ge N, Drevet J, Boyer N (1998) Molecular cloning and characterization of tomato cDNAs encoding glutathione peroxidase-like proteins. Eur J Biochem 253: 445–451 Dickman MB, Park YK, Oltersdorf T, Li W, Clemente T, French R (2001) Abrogation of disease development in plants expressing animal antiapoptotic genes. Proc Natl Acad Sci USA 98: 6957–6962 Dickman MB, Reed JC (2003) Paradigms for programmed cell death in animals and plants. In J Gray, ed, Programmed Cell Death in Plants. Blackwell, Oxford, pp 26–43 el-Saadani M, Esterbauer H, el-Sayed M, Goher M, Nassar AY, Jurgens G (1989) A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J Lipid Res 30: 627–630 Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8: 978–984 Epp O, Ladenstein R, Wendel A (1983) The refined structure of the selenoenzyme glutathione peroxidase at 0.2-nm resolution. Eur J Biochem 133: 51–69
1640
Frohlich KU, Madeo F (2000) Apoptosis in yeast—a monocellular organism exhibits altruistic behaviour. FEBS Lett 473: 6–9 Gladyshev VN, Jeang KT, Stadtman TC (1996) Selenocysteine, identified as the penultimate C-terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene. Proc Natl Acad Sci USA 93: 6146–6151 Gueta-Dahan Y, Yaniv Z, Zilinskas BA, Ben-Hayyim G (1997) Salt and oxidative stress: similar and specific responses and their relation to salt tolerance in citrus. Planta 203: 460–469 Havel L, Durzan DJ (1996) Apoptosis in plants. Bot Acta 109: 268–277 Hazebrouck S, Camoin L, Faltin Z, Strosberg AD, Eshdat Y (2000) Substituting selenocysteine for catalytic cysteine 41 enhances enzymatic activity of plant phospholipid hydroperoxide glutathione peroxidase expressed in Escherichia coli. J Biol Chem 275: 28715–28721 Herbette S, Lenne C, Leblanc N, Julien JL, Drevet JR, Roeckel-Drevet P (2002) Two GPX-like proteins from Lycopersicon esculentum and Helianthus annus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities. Eur J Biochem 269: 2414–2420 Holland D, Ben-Hayyim G, Faltin Z, Camoin L, Strosberg AD, Eshdat Y (1993) Molecular characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases. Plant Mol Biol 21: 923–927 Jazwinski SM (1993) The genetics of aging in the yeast Saccharomyces cerevisiae. Genetica 91: 35–51 Jin C, Reed JC (2002) Yeast and apoptosis. Nat Rev Mol Cell Biol 3: 453–459 Jones JDG, Shlumukov L, Carland FJ, Scofield S, Bishop G, Harrison K (1992) Effective vectors for tranformation, expression of heterologous genes and assaying transposon excision in transgenic plants. Transgenic Res 1: 285–297 Jung BG, Lee KO, Lee SS, Chi YH, Jang HH, Kang SS, Lee K, Lim D, Yoon SC, Yun DJ, et al (2002) A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase. J Biol Chem 277: 12572–12578 Kampranis SC, Damianova R, Atallah M, Toby G, Kondi G, Tsichlis PN, Makris AM (2000) A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast. J Biol Chem 275: 29207–29216 Kawai M, Pan L, Reed JC, Uchimiya H (1999) Evolutionally conserved plant homologue of the Bax inhibitor-1(BI-1) gene capable of suppressing Bax-induced cell death in yeast. FEBS Lett 464: 143–147 Kawai-Yamada M, Jin L, Yoshinaga K, Hirata A, Uchimiya H (2001) Mammalian Bax-induced plant cell death can be down-regulated by overexpression of Arabidopsis Bax Inhibitor-1 (AtBI-1). Proc Natl Acad Sci USA 98: 12295–12300 Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239–257 Kuzniak E, Sklodowska M (2004) The effect of Botrytis cinerea infection on the antioxidant profile of mitochondria from tomato leaves. J Exp Bot 55: 605–612 Lacomme C, Santa Cruz S (1999) Bax-induced cell death in tobacco is similar to the hypersensitive response. Proc Natl Acad Sci USA 96: 7956–7961 Lee AC, Fenster BE, Ito H, Takeda K, Bae NS, Hirai T, Yu ZX, Ferrans VJ, Howard BH, Finkel T (1999) Ras proteins induce senescence by altering the intracellular levels of reactive oxygen species. J Biol Chem 274: 7936–7940 Li W, Liu J, Zhao N (2001) Cloning and characterization of a PHGPX gene from Momordica. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Jin Zhan 28: 908–911 Madeo F, Engelhardt S, Herker E, Lehmann N, Maldener C, Proksch A, Wissing S, Frohlich KU (2002) Apoptosis in yeast: a new model system with applications in cell biology and medicine. Curr Genet 41: 208–216 Madeo F, Frohlich E, Ligr M, Grey M, Sigrist SJ, Wolf DH, Frohlich KU (1999) Oxygen stress: a regulator of apoptosis in yeast. J Cell Biol 145: 757–767 Maiorino M, Gregolin C, Ursini F (1990) Phospholipid hydroperoxide glutathione peroxidase. Methods Enzymol 186: 448–457 McCabe PF, Valentine TA, Forsberg LS, Pennell RI (1997) Soluble signals from cells identified at the cell wall establish a developmental pathway in carrot. Plant Cell 9: 2225–2241
Plant Physiol. Vol. 135, 2004
Tomato Phospholipid Peroxidase Inhibits Cell Death
McNellis TW, Mudgett MB, Li K, Aoyama T, Horvath D, Chua NH, Staskawicz BJ (1998) Glucocorticoid-inducible expression of a bacterial avirulence gene in transgenic Arabidopsis induces hypersensitive cell death. Plant J 14: 247–257 Mitsuhara I, Malik KA, Miura M, Ohashi Y (1999) Animal cell-death suppressors Bcl-x(L) and Ced-9 inhibit cell death in tobacco plants. Curr Biol 9: 775–778 Moon H, Baek D, Lee B, Prasad DT, Lee SY, Cho MJ, Lim CO, Choi MS, Bahk J, Kim MO, et al (2002) Soybean ascorbate peroxidase suppresses Bax-induced apoptosis in yeast by inhibiting oxygen radical generation. Biochem Biophys Res Commun 290: 457–462 Navarre DA, Wolpert TJ (1999) Victorin induction of an poptotic/ senescence-like response in oats. Plant Cell 11: 237–249 Nomura K, Imai H, Koumura T, Arai M, Nakagawa Y (1999) Mitochondrial phospholipid hydroperoxide glutathione peroxidase suppresses apoptosis mediated by a mitochondrial death pathway. J Biol Chem 274: 29294–29302 Nomura K, Imai H, Koumura T, Kobayashi T, Nakagawa Y (2000) Mitochondrial phospholipid hydroperoxide glutathione peroxidase inhibits the release of cytochrome c from mitochondria by suppressing the peroxidation of cardiolipin in hypoglycaemia-induced apoptosis. Biochem J 351: 183–193 Reuber TL, Ausubel FM (1996) Isolation of Arabidopsis genes that differentiate between resistance responses mediated by the RPS2 and RPM1 disease resistance genes. Plant Cell 8: 241–249 Rodriguez Milla MA, Maurer A, Huete Rodriguez A, Gustafson JP (2003) Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses through diverse signaling pathways. Plant J 36: 602–615 Roxas VP, Lodhi SA, Garrett DK, Mahan JR, Allen RD (2000) Stress tolerance in transgenic tobacco seedlings that overexpress glutathione S-transferase/glutathione peroxidase. Plant Cell Physiol 41: 1229–1234 Roxas VP, Smith RK, Allen ER, Allen RD (1997) Overexpression of glutathione S-transferase/glutathione peroxidase enhances the growth of transgenic tobacco seedlings during stress. Nat Biotechnol 15: 988–991 Samali A, Holmberg CI, Sistonen L, Orrenius S (1999) Thermotolerance and cell death are distinct cellular responses to stress dependence on heat shock proteins. FEBS Lett 46: 306–310 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Labora-
Plant Physiol. Vol. 135, 2004
tory Manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Serrano M, Blasco MA (2001) Putting the stress on senescence. Curr Opin Cell Biol 13: 748–753 Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A (1999) The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11: 431–444 Stadtman TC (1996) Selenocysteine. Annu Rev Biochem 65: 83–100 Takken FL, Luderer R, Gabriels SH, Westerink N, Lu R, de Wit PJ, Joosten MH (2000) A functional cloning strategy, based on a binary PVXexpression vector, to isolate HR-inducing cDNAs of plant pathogens. Plant J 24: 275–283 Tao W, Walke DW, Morgan JI (1999) Oligomerized Ced-4 kills budding yeast through a caspase-independent mechanism. Biochem Biophys Res Comm 260: 799–805 Tiedmann AV (1997) Evidence for the induction of active oxygen species in the induction of host cell death during infection of bean leaves with Botrytis cinerea. Physiol Mol Plant Pathol 50: 151–166 Ursini F, Maiorino M, Gregolin C (1985) The selenoenzyme phospholipid hydroperoxide glutathione peroxidase. Biochim Biophys Acta 839: 62–70 Vaux DL, Strasser A (1996) The molecular biology of apoptosis. Proc Natl Acad Sci USA 93: 2239–2244 Vaux DL, Weissman IL, Kim SK (1992) Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2. Science 258: 1955–1957 Vaux EC, Wood SM, Cockman ME, Nicholls LG, Yeates KM, Pugh CW, Maxwell PH, Ratcliffe PJ (2001) Selection of mutant CHO cells with constitutive activation of the HIF system and inactivation of the von Hippel-Lindau tumor suppressor. J Biol Chem 276: 44323–44330 Wang W, Jones C, Ciacci-Zanella J, Holt T, Gilchrist DG, Dickman MB (1996) Fumonisins and Alternaria alternata lycopersici toxins: sphinganine analog mycotoxins induce apoptosis in monkey kidney cells. Proc Natl Acad Sci USA 93: 3461–3465 White JL, Kaper JM (1989) A simple method for detection of viral satellite RNAs in small plant tissue samples. J Virol Methods 23: 83–94 Xu Q, Reed JC (1998) Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast. Mol Cell 1: 337–346 Zha H, Fisk HA, Yaffe MP, Mahajan N, Herman B, Reed JC (1996) Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells. Mol Cell Biol 16: 6494–6508
1641
