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Plant Physiol. Sep 2006; 142(1): 193–206.
PMCID: PMC1557616

Polyamine Oxidase Is One of the Key Elements for Oxidative Burst to Induce Programmed Cell Death in Tobacco Cultured Cells1

Abstract

Programmed cell death plays a critical role during the hypersensitive response in the plant defense system. One of components that triggers it is hydrogen peroxide, which is generated through multiple pathways. One example is proposed to be polyamine oxidation, but direct evidence for this has been limited. In this article, we investigated relationships among polyamine oxidase, hydrogen peroxide, and programmed cell death using a model system constituted of tobacco (Nicotiana tabacum) cultured cell and its elicitor, cryptogein. When cultured cells were treated with cryptogein, programmed cell death occurred with a distinct pattern of DNA degradation. The level of hydrogen peroxide was simultaneously increased, along with polyamine oxidase activity in apoplast. With the same treatment in the presence of α-difluoromethyl-Orn, an inhibitor of polyamine biosynthesis, production of hydrogen peroxide was suppressed and programmed cell death did not occur. A gene encoding a tobacco polyamine oxidase that resides in the apoplast was isolated and used to construct RNAi transgenic cell lines. When these lines were treated with cryptogein, polyamines were not degraded but secreted into culture medium and hydrogen peroxide was scarcely produced, with a concomitant suppression of cell death. Activities of mitogen-activated protein kinases (wound- and salicylic acid-induced protein kinases) were also suppressed, indicating that phosphorylation cascade is involved in polyamine oxidation-derived cell death. These results suggest that polyamine oxidase is a key element for the oxidative burst, which is essential for induction of programmed cell death, and that mitogen-activated protein kinase is one of the factors that mediate this pathway.

Plant disease resistance is initiated by specific recognition of pathogen-derived molecules by plant cells (Yang et al., 1997). Upon pathogen attack, necrotic lesions are formed at the site of pathogen entry, resulting in prevention of further spread of disease, this phenomenon being called the hypersensitive response (HR). One of the earliest events that occurs during HR is the production of reactive oxygen intermediates (ROI), this is known as the oxidative burst. ROI mainly include superoxide, hydroxyl radicals, and hydrogen peroxide, and play a critical role in triggering and maintaining the HR by directly attacking pathogens, serving as substrates for lignification and cross-linking of extensin, mediating signal transduction pathways, and inducing programmed cell death (Levine et al., 1994; Bestwick et al., 1997; Thordal et al., 1997). As sources of ROI, pathways catalyzed by several enzymes have been proposed, including NADPH oxidase (Keller et al., 1998), peroxidase (Bolwell and Wojitaszek, 1997), oxalate oxidase (Hu et al., 2003), copper-containing amine oxidases (Allan and Fluhr, 1997), and polyamine oxidases (Yoda et al., 2003). Which of them constitutes the main source of ROI in particular cases remains to be determined.

It is well documented that generation of ROI exhibits two phases during HR, the rapid and transient phase I and the late and persistent phase II (Grant, 1997; Lamb and Dixon, 1997; Grant and Loake, 2000). This suggests that multiple sources of ROI might be present. NADPH oxidase located in plasma membranes may be the most significant source of ROI, being activated immediately after pathogen recognition (Keller et al., 1998). In apoplasts, oxygen is converted by NADPH oxidase into superoxide, which spontaneously dismutates to give another active oxygen intermediate, hydrogen peroxide (Bestwick et al., 1997). Polyamines have also been proposed as important substrates for hydrogen peroxide production, being degraded by copper-containing amine oxidases or polyamine oxidases (Šebela et al., 2001; Yoda et al., 2003). In fact, enzymatic activities of polyamine metabolism are reported to increase in barley (Hordeum vulgare) seedlings during HR in response to powdery mildew (Cowley and Walters, 2002). In animal cells, polyamines can be oxidized by polyamine oxidases to yield hydrogen peroxide, which eventually induces programmed cell death (Ha et al., 1997; Lindsay and Wallace, 1999; Facchiano et al., 2001). It is therefore highly probable that enzymatic degradation of polyamines also directly contributes to ROI production and programmed cell death in plants.

Using Tobacco (Nicotiana tabacum) mosaic virus (TMV) and intact tobacco cultivars carrying the resistant (N) gene, we previously demonstrated that polyamines are indeed one of the sources of hydrogen peroxide during HR (Yoda et al., 2003). We found that, upon HR induction, polyamines accumulate in apoplasts and are degraded, resulting in the production of hydrogen peroxide. Since polyamine oxidase appeared to play a key role in this system, we here isolated a gene encoding a polyamine oxidase, and characterized its function using tobacco cultured cells (Bright-Yellow 2 [BY2]) and a non-race-specific proteinaceous elicitor, cryptogein. Cryptogein is originally derived from a fungal pathogen, Phytophthora cryptogea, which induces an HR-like response in tobacco, including the oxidative burst, nitric oxide production, and hypersensitive cell death (Ponchet et al., 1999; Lamotte et al., 2004). The advantage of this system is not only that HR-like response can be synchronously induced, but also that production of ROI clearly precedes HR-like cell death (Kadota et al., 2004).

During activation of plant defense responses, mitogen-activated protein kinases (MAPKs) play crucial roles in signaling pathways. In tobacco plants carrying the N gene, TMV infection leads to activation of two MAPKs, salicylic acid (SA)-induced protein kinase (SIPK), and wound-induced protein kinase (WIPK; Zhang and Klessig, 1998). The activation is also observed when tobacco plants or tobacco cultured cells are treated with non-race-specific elicitors, including fungal cell wall-derived elicitors, INF1, harpin, and cryptogein (Suzuki et al., 1999; Zhang et al., 2000; Sharma et al., 2003; Samuel et al., 2005). Their orthologs in other plant species have also been implicated in defense signaling (Zhang and Klessig, 2001). In all cases, the activation of MAPKs precedes and correlates with the onset of HR-like cell death.

Here we provide evidence that polyamine oxidase is responsible for generation of the oxidative burst, resulting in programmed cell death, and that MAPK is one of the signaling components in the polyamine oxidase-dependent defense response.

RESULTS

Programmed Cell Death Induced by Cryptogein

To determine whether cryptogein-induced cell death is programmed, patterns of DNA cleavage were analyzed. BY2 cells were treated with cryptogein for 24 h and DNA cleavage was examined with gel electrophoresis. The results showed a specific DNA laddering pattern, consisting of multiples of about 180 bp (Fig. 1A), which was not evident with DNA from untreated cells (Fig. 1A). In situ DNA cleavage was then examined by subjecting cryptogein-treated cells to 3′-end DNA labeling with terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL assay). Bright fluorescent signals were evident in nuclei, indicating DNA to be fragmented (Fig. 1B). Untreated cells were not labeled (Fig. 1B). The results are consistent with the case of animal cells undergoing apoptosis, showing a characteristic laddering pattern with nucleosome-sized DNA fragments and positive TUNEL staining, and strongly suggesting that programmed cell death indeed takes place in BY2 cells upon cryptogein treatment.

Figure 1.
Programmed cell death induced by cryptogein. A, DNA laddering pattern. A 5-μg aliquot of DNA extracted from BY2 cells treated with 250 nm cryptogein for 0 or 24 h was fractionated on a 2% agarose gel and visualized with ethidium bromide staining. ...

Suppression of Programmed Cell Death

To establish an experimental system in which programmed cell death is controlled, we first examined the effect of an elicitor, cryptogein, and inhibitors of polyamine synthetic pathways. α-Difluoromethyl-Arg, an irreversible inhibitor of Arg decarboxylase, which catalyzes conversion of Arg into agmatine, did not affect cryptogein-induced cell death (data not shown). Cyclohexylamine, an inhibitor of spermidine synthase, induced cell death in a dose-dependent manner, probably due to ectopic accumulation of putrescine (data not shown). Neither inhibitor affected cell viability by themselves (data not shown). Since α-difluoromethyl-Orn (DFMO), an irreversible inhibitor of Orn decarboxylase, clearly suppressed hypersensitive cell death in TMV-infected tobacco leaves (Yoda et al., 2003), we further examined its effects on BY2 cell growth. Experimentally, BY2 cells were treated with or without cryptogein in the presence or absence of DFMO. Effects of cryptogein on BY2 cell growth were obvious, with almost complete suppression of cell proliferation (Fig. 2A). In the presence of DFMO, however, the rate of cell growth recovered to the control level. DFMO itself did not affect the cell growth up to 5 d after treatment (Fig. 2A). When BY2 cells were cultivated in the absence of drugs, the death rate was low (approximately 3%) and this level remained constant for up to 48 h (Fig. 2B). In the presence of 50 nm cryptogein, it significantly increased after 12 h and reached over 30% after 24 h (Fig. 2B). However, this cryptogein-induced cell death was largely suppressed in the presence of DFMO, which again did not show any effects on cell death by itself (Fig. 2B). When directly visualized by Evans blue staining, cell death was found to have occurred in approximately 30% of the total cell population 48 h after elicitation. Treatment with cryptogein in the presence of DFMO resulted in far fewer dead cells, while DFMO itself did not affect cell viability (Fig. 2C). Since these results were suggestive of an involvement of polyamines in HR-like cell death, effects of exogenously applied putrecine, spermidine, and spermine were then examined. Suppression of cryptogein-induced cell death by DFMO was reversed by the addition of spermidine or spermine, but not putrescine (Fig. 2D). Based on these observations, DFMO was employed in further experiments.

Figure 2.
Inhibition and rescue of cryptogein-induced cell death. A, Effects of DFMO on cell growth. BY2 cells in the absence of drugs (white circles) or in the presence of 50 nm cryptogein (black circles), both cryptogein and 2 mm DFMO (white triangles), or 2 ...

Production of ROI

Since polyamines are degraded to produce hydrogen peroxide, we next examined the amount of hydrogen peroxide released during elicitation. When BY2 cells were sampled every 15 min after cryptogein treatment, hydrogen peroxide was detected as early as within the first 15 min (Fig. 3A). The amount of hydrogen peroxide increased thereafter, reaching a maximal level at 6 h, and then gradually declined over the next 24 h (Fig. 3A). When DFMO was applied prior to cryptogein treatment, the induction profile of hydrogen peroxide greatly changed, showing an almost complete inhibition 3 h after elicitation (Fig. 3A). When spermidine was added to the medium in the presence of both cryptogein and DFMO, the inhibition was mitigated throughout the examined period (Fig. 3B). These results clearly indicated that the majority of the oxidative burst in the late phase is generated through polyamine degradation.

Figure 3.
Time-course analyses of accumulation of hydrogen peroxide and superoxide during elicitation. A, Effects of DFMO on hydrogen peroxide production. BY2 cells were cultured in the presence of 50 nm cryptogein (white circles), both cryptogein and 2 mm DFMO ...

NADPH oxidase has long been thought to contribute to the oxidative burst, generating superoxide that is then converted into hydrogen peroxide by superoxide dismutase. Subsequently, we directly estimated the amount of superoxide, and found effective generation in cells treated with cryptogein throughout the examined period, but particularly during the initial 4 h (Fig. 3C). Its level reached a maximum 4 h after elicitation and declined thereafter. When cells were treated with cryptogein in the presence of DFMO, the generation of superoxide was not greatly influenced, with only a slight reduction (Fig. 3C). In contrast, addition of superoxide dismutase to the sample completely abolished its production (Fig. 3C). These results suggest that at least two systems are involved in hydrogen peroxide production during cryptogein-induced programmed cell death, one featuring NADPH oxidase and the other polyamine degradation.

Polyamine Oxidation

Polyamine contents were directly estimated after elicitation. When BY2 cells were treated with cryptogein, the level of spermidine in apoplasts gradually increased, showing, for example, 13 nmol and 71 nmol g−1 fresh weight at the 9- and 48-h time points, respectively (Fig. 4A). Since the spermidine level in untreated samples was approximately 3.5 nmol g−1 fresh weight, the increase after 48 h was 20-fold the control values. Accumulation of spermine was less distinct, being only detectable 24 h after elicitation, and increasing up to 33 nmol g−1 fresh weight 48 h later (data not shown). The level of putrescine was only 2-fold the control value 48 h after elicitation (data not shown). In the presence of DFMO, neither of the polyamines was elevated (Fig. 4A). Polyamine oxidase activity in apoplasts was then examined. To this end, the purity of apoplastic fluids was first evaluated by immunoblot analysis using antibodies against phosphoenolpyruvate carboxylase (PEPC), which is used as the cytoplasm marker. The protein was clearly absent from apoplastic fraction, indicating that examined apoplastic fluids were not contaminated with cytoplasmic contents (Fig. 4B). When spermidine was employed as the substrate for this fraction, the activity was detectable but very low in untreated control cells throughout the examined period (Fig. 4C). On elicitation, the activity clearly began to increase within 6 h, and reached a maximum of 10-fold the background level after 24 h (Fig. 4C). However, when DFMO was applied together with cryptogein to the samples, the activity was markedly reduced throughout the time period (Fig. 4C), suggesting it to be regulated by the available substrate concentration. When spermine was used as the substrate, similar activity profiles were obtained, whereas no activity was found with putrescine throughout the examined period (data not shown). The identity of polyamine oxidase was confirmed by activity inhibition by guazatine, a competitive inhibitor of polyamine oxidase (Binda et al., 2001). When being applied 10- and 50-fold excess of the substrate, the activity reduced to 30% and 70% of the control, respectively (Fig. 4D). Effects of guazatine on cell death were finally examined. While guazatine itself was not detrimental to cell growth, cryptogein-induced cell death was largely mitigated by this chemical, clearly indicating that polyamine oxidase was critical for cell death induction (Fig. 4E). That polyamine oxidase in apoplastic fluids was an inherent resident in apoplasts was confirmed with two additional experiments: First, the activity in media was found to be low throughout the experimental period (data not shown), and second, the activity was not detectable in crude extracts of remaining cells after apoplast extraction (data not shown).

Figure 4.
Accumulation of polyamines and polyamine oxidase activity. A, Cells were treated with 50 nm cryptogein for the indicated time periods in the absence (black circles) or presence (white triangles) of 2 mm DFMO. The controls were intact untreated cells (white ...

Scavenging of ROI

Hydrogen peroxide reached a maximum 6 to approximately 7 h after elicitation and declined thereafter (Fig. 3A), while polyamine oxidase activity, which was constantly detected at relatively low levels, only began to increase at 6 h and remained high up to 24 h after elicitation (Fig. 4B). This time gap could be due to the activation of scavenger systems for ROI, including catalase and ascorbate peroxidase enzymes. Total hydrogen peroxide scavenging activity in cells was kinetically measured after cryptogein treatment (Fig. 5). The results indicated that BY2 cells treated with cryptogein exhibit hydrogen peroxide degradation, the activity varying at different time points after elicitation. When relative activities obtained at a fixed incubation time (25 min) were plotted, it was evident that the activity began to decline 3 h after elicitation, reaching a minimum at 12 h (Fig. 5, insert). The activity then was restored to the initial level thereafter. These results suggest that accumulation of hydrogen peroxide during the initial 6 h after elicitation is partly due to a temporary decline of ROI scavenger activity, and that a high level of hydrogen peroxide degradation beyond 12 h is attributable to a restoration of scavenger activity. Thus, there is a clear inverse relationship between the production (Fig. 3A) and degradation (Fig. 5) activities of hydrogen peroxide during the HR.

Figure 5.
Scavenging activity of ROI during the HR. BY2 cells treated with 50 nm cryptogein for the indicated time periods were collected by filtration and resuspended in fresh medium containing 10 mm hydrogen peroxide. The concentration of hydrogen peroxide was ...

Isolation of a Polyamine Oxidase Gene

The above described observations point to polyamine oxidase(s) playing a critical role in hydrogen peroxide production during elicitation, and led us to further analyze its function. However, since the tobacco gene encoding apoplast-resident polyamine oxidase was not known, we isolated a corresponding cDNA from a library derived from TMV-infected tobacco leaves (cv Xanthi nc). The clone finally obtained consists of 1,917 bp and encodes a putative protein with 495 amino acids, showing similarity to polyamine oxidase from tomato (Lycopersicon esculentum; 87%) and from Arabidopsis (Arabidopsis thaliana; 76%; Fig. 6A). The gene, designated as Nicotiana tabacum polyamine oxidase (NtPAO), was found to be constantly expressed in BY2 cells under normal culture conditions, and cryptogein treatment did not stimulate transcript accumulation (data not shown). The predicted molecular mass of mature NtPAO is 53.6 kD, which is consistent with that calculated from a gel filtration assay (Yoda et al., 2003). Protein Localization Sites version 6.4 suggested that the initial 22 amino acids in the N terminus were a signal peptide for apoplast (Fig. 6A).

Figure 6.
Properties of NtPAO. A, Amino acid sequence alignment of NtPAO with related proteins. NtPAO (accession no. AB200262) was compared with LePAO (TC119169) ...

To confirm the enzymatic activity, glutathione S-transferase (GST)-fused NtPAO protein, which lacks the initial 22 amino acids, was expressed in Escherichia coli and purified through glutathione-Sepharose 4B affinity column (Fig. 6B). Polyamine oxidase is a FAD-dependent enzyme, which catalyzes polyamine oxidation coupling with FAD reduction (Polticelli et al., 2005). Polyamine oxidase activity was then measured with different substrates in the presence or absence of FAD (Fig. 6C). A high activity was detected with spermidine and spermine, while no activity was found with putrescine (Fig. 6C). In the absence of FAD, the enzyme showed no activity even with the best substrate, spermidine, indicating the isolated NtPAO to be flavoprotein. To substantiate its subcellular localization, a fusion protein was constructed, by fusing GFP to the C terminus of NtPAO, and transiently expressed in onion (Allium cepa) epidermal cells. While fluorescence from BY2 cells expressing only GFP was observed in cytosol and nucleus, in cells expressing the fusion protein it was detected in areas around cells (Fig. 6D), indicating that NtPAO is localized in apoplasts, consistent with our observation of polyamine oxidase activity in apoplastic fluids (Fig. 4, B and C). The results also suggested that NtPAO is the very enzyme that is involved in polyamine degradation during elicitation.

Construction of Polyamine Oxidase RNAi Lines

The physiological function of NtPAO was examined using transgenic BY2 cells, in which its activity was suppressed by the RNAi method. Among five transformants, two lines, numbers 1 and 2, showed the activity of undetectable level 12 h after elicitation (Fig. 7A). This indicated that the RNAi construct was efficient enough for further experiments. Using these RNAi lines, we subsequently examined hydrogen peroxide production and found that, in both lines, it hardly accumulated after elicitation in contrast to wild-type cells (Fig. 7B), this being particularly distinct in line 2. It is notable that, even in line 2, a detectable level of hydrogen peroxide was observed 2 h after elicitation. Cryptogein-induced programmed cell death was also suppressed in transgenic line 2 in comparison with control cells (Fig. 7C). While up to 25% of the wild-type cells were dead after 6 h elicitation, dead cells constituted only 7% of the transgenic population. Such a difference was also observed with 12 h elicitation. Cell death was visually confirmed by microscopic observation, which clearly showed almost no dead cells in transgenic lines, whereas 27% were dead in the control case (Fig. 7D). The level of polyamines was directly estimated in wild-type and line 2 cells after elicitation. Its level in apoplasts is less than half in line 2 than in wild-type cells at 24 h after elicitation (Fig. 7E). However, spermidine level in culture media was found to be 14-fold higher in line 2 than in wild-type cells at 24 h after elicitation (Fig. 7E), suggesting that spermidine was actively secreted out of cells in the former. Putrescine level was also high in line 2 culture media, but spermine was undetectable in both cells (data not shown). Finally, the effect of secreted spermidine on NADPH oxidase activity in line 2 was examined. Experimentally, wild-type and line 2 cells were treated with cryptogein and subjected to direct estimation of the enzymatic activity according to the published method (Sagi and Fluhr, 2001). However, results somehow fluctuated, perhaps due to different materials. Consequently, the production of superoxide, the direct product of NADPH oxidase reaction, was measured and found to be less than half in line 2 than in wild-type cells 5 h after elicitation (Fig. 7F).

Figure 7.
Analyses of polyamine oxidase RNAi transgenic cells. A, Activity assay of polyamine oxidase. Wild-type (white bars) and transgenic (nos. 1 and 2, black and hatched bars, respectively) BY2 cells were treated with cryptogein and enzymatic activity was determined ...

Effects on HR-Related Gene Expression and MAPK Activity

The observed phenotype of transgenic cells suggested altered defense response. To verify this idea, nontransformed wild-type and transgenic line 2 cells were treated with cryptogein, and expression of HR-marker genes was examined by RNA-blot hybridization. Transcripts for HSR203J (for hypersensitivity related) and HIN1 (for harpin induced) were observed in both wild-type and transgenic line 2 cells at 3 h later. However, the induction level was considerably lower and more prolonged in the latter than in the former (Fig. 8A). In contrast, transcripts for pathogenesis-related (PR)-1b were induced at much higher and faster rate in transgenic line 2 than in wild-type cells (Fig. 8B). This could be attributed to the elevated spermidine concentration in culture medium of transgenic cells (Fig. 7E), secondarily stimulating its expression (Yamakawa et al., 1998).

Figure 8.
Change of HR-related gene expression in RNAi transgenic cells. Total RNA was isolated from wild-type (WT) and transgenic (no. 2) cells, which were treated with cryptogein for indicated time periods. RNA gel-blot analysis was conducted with indicated probes ...

It was reported that two MAPKs, SIPK and WIPK, are enzymatically activated by spermine (Takahashi et al., 2003). When MAPK activity was evaluated by an in-gel kinase assay using myelin basic protein as the substrate, protein kinase activity at 44 and 46 kD was apparently induced in nontransformed BY2 cells treated with cryptogein (Fig. 9A), suggesting correspondence with SIPK and WIPK, respectively (Jonak et al., 2002). SIPK was activated as early as 30 min after elicitation, and WIPK after 3 h, elevation then being maintained for at least 8 h (Fig. 9A). In RNAi line 2, however, activities were weak and transient, showing a maximum only at 3 h. In BY2 cells treated with cryptogein in the presence of DFMO, suppression of their activity was even more evident than in RNAi 2 cells, showing SIPK only at 3 h, and almost no induction of WIPK (Fig. 9A). Finally, effects of polyamines on MAPK activity were examined. In wild-type cells, spermidine markedly activated SIPK but not WIPK 6 h after treatment (Fig. 9B). In transgenic RNAi 2 cells, the same induction profile was also observed, but the level was much less than that of the controls (Fig. 9B). Neither spermine nor putrescine was effective under our experimental conditions (data not shown).

Figure 9.
Suppression of MAPK activity. A, In-gel kinase assays for MAPK activity. Wild-type BY2 cells were treated with cryptogein in the absence (WT) or presence (DFMO) of DFMO for the indicated time periods. Transgenic line number 2 cells (#2) were also ...

DISCUSSION

ROI is a crucial element for induction of programmed cell death during HR and HR-like responses (Hammond-Kosack and Jones, 1996; Lamb and Dixon, 1997). Since ROI can be detected as early as within minutes upon the onset of HR, its production is thought to be one key factor in the defense response. The source, however, may vary, although NADPH oxidase is considered to be a major contributor because of its similarity with neutrophil enzymes in mammals (Groom et al., 1996; Keller et al., 1998). In a previous investigation with intact tobacco leaves and TMV, we demonstrated that genes encoding enzymes involved in polyamine biosynthesis are markedly up-regulated upon the onset of HR (Yoda et al., 2003). Subsequently, polyamines were found to accumulate in apoplasts of infected leaves and to be degraded by polyamine oxidase, releasing hydrogen peroxide that causes hypersensitive cell death (Yoda et al., 2003). This raised questions as to the extent of the overall contribution of polyamine-derived hydrogen peroxide to hypersensitive cell death.

Temporal Features

To address this question, we developed the present system of tobacco cultured cells exposed to cryptogein. Trials established that BY2 cells, like tobacco cultured cells derived from tobacco cv Xanthi nc (Binet et al., 2001), can be efficiently stimulated to undergo programmed cell death by cryptogein. Time-course analyses using this system indeed showed clear spatial and temporal accumulation and degradation of components involved in hydrogen peroxide metabolism (Fig. 10). Accumulation of polyamines proved variable, with a gradual increase of spermidine but almost none of spermine before 24 h. Polyamine oxidase activity was constitutively detectable at a low level, but markedly increased after elicitation. Hydrogen peroxide production occurred as early as 15 min after elicitation, reaching a maximal level at 6 h and then declined. This feature was inversely well correlated with scavenger activity of hydrogen peroxide. The signal transducers, SIPK and WIPK, were activated within 30 min and 3 h, respectively. Hypersensitive cell death was apparent 6 h later. Based on these findings we propose that upon elicitation, polyamines are simultaneously produced and degraded, releasing hydrogen peroxide, which activates signal transduction pathways including MAPKs to induce programmed cell death. Although the overall feature of polyamine-dependent programmed cell death can be explained by this idea, some comments are needed as to relationships among the factors involved.

Figure 10.
Kinetic outline of factors involved in polyamine-dependent cell death. The vertical axis indicates relative values for the indicated event, and the horizontal axis the time elapsed after onset of the HR. Spd and Spm stand for spermidine and spermine, ...

Accumulation of polyamines began after the onset of oxidative burst. This can be explained by their simultaneous production and degradation, as seen from a vast accumulation of spermidine at relatively early stage in culture fluid of the RNAi line, in which polyamine oxidase activity is suppressed. Suppression of oxidative burst in both DFMO-treated cells and polyamine oxidase RNAi cell lines also supports this idea. In wild-type plants, perhaps polyamine synthesis is well concerted with oxidation, resulting in apparently no accumulation during the oxidative burst occurring at the first 12 h. Polyamines are gradually accumulated thereafter despite the increase of polyamine oxidase. We have no clear explanation for this, but several cases are conceivable; for example, differential rate between production and degradation, masking polyamine oxidase activity in vivo, and protection of polyamines by other components.

Hydrogen peroxide level declines 6 h after elicitation, while polyamines and polyamine oxidase activity constantly increase. This can be partly due to scavenging activity of ROI, which is transiently decreased during the first 12 h after elicitation and increased to the basal level thereafter. Hydrogen peroxide produced later than 12 h might be erased by this recovered scavenging activity. Another possibility of its apparent decline at the late stage is consumption of hydrogen peroxide for repair process. For example, during defense response, ROI is consumed to strengthen cell wall for physiological barrier against pathogens via oxidative cross-linking of glycoproteins (Bradley et al., 1992) and lignification from oxidative polymerization of monolignols (Kawasaki et al., 2006).

Spermidine was significantly secreted into medium and not retained in apoplasts of RNAi cells. Since the activity of hydrogen peroxide production is suppressed in RNAi transgenic lines, cell wall restoration might be low as discussed above. This may cause a loose structure of cell wall through which polyamines could leak into the medium as observed.

Exogenously applied putrescine, a direct reaction product of Orn decarboxylase that is inhibited by DFMO, does not rescue DFMO-dependent suppression of cell death. This suggests that putrescine is not directly involved in polyamine-dependent cell death. Since its accumulation was less evident in comparison with other polyamines, putrescine may serve as the intermediate for polyamine synthesis in the present system. Alternatively, since putrescine prevents programmed cell death syndrome (Papadakis and Roubelakis-Angelakis, 2005), it may also function as a negative regulator to limit the propagation of programmed cell death.

As spermine is known as an inducer for PR gene expression (Yamakawa et al., 1998) and as an activator for MAPKs in tobacco plants (Takahashi et al., 2003), its role in our system should be considered. In the present study, spermine began to accumulate at least 24 h after elicitation, when expression of PR-1b was fully detectable. However, MAPKs are activated within 30 min with a gradual increase during hours after elicitation, when spermine accumulation is not observed. This suggests that spermine is not involved in modulating the MAPK activities by itself. It is conceivable that spermine serves as the substrate for polyamine oxidase at the early stage, and as the activator for such as PR genes at the late stage of the HR, although the possibility is not excluded that elicitation renders BY2 cells more sensitive to spermine, resulting in activation of MAPKs before apparent accumulation of spermine takes place.

Source for Oxidative Burst

Regarding the two phases of the oxidative burst (Grant, 1997; Lamb and Dixon, 1997; Grant and Loake, 2000), the amount of ROI in the prolonged phase II is markedly more abundant than in phase I (Draper, 1997). At present, the biological function of ROI in each phase is not completely understood. However, in phase I it may not always correlate with plant disease resistance, whereas the link with the defense response appears more absolute in phase II (Baker and Orlandi, 1995). Analyses have suggested that ROI in phase I is dependent on NADPH oxidase based on the following observations: First, suppression of the gene encoding gp91phox homologs (rboh), one component of the NADPH oxidase complex, resulted in a disruption of both phases in Arabidopsis (Torres et al., 2002) and tobacco (Simon-Plas et al., 2002); second, oxidative burst is sensitive to diphenylene iodonium, an inhibitor of flavin-containing oxidase including NADPH oxidase (Rustérucci et al., 1996; Simon-Plas et al., 1997); third, a tobacco NtrbohD was rapidly and transiently expressed in cryptogein-treated cultured cells (Simon-Plas et al., 2002), this being consistent with our present observation of rapid and transient production of superoxide. Further analysis recently indicated that spermine and spermidine, but not putrescine, inhibit NADPH oxidase activity in a concentration-dependent manner (Papadakis and Roubelakis-Angelakis, 2005). Our present results agree with these observations and suggest that observed inability of hydrogen peroxide production in RNAi cells was partly caused by the inhibition of NADPH oxidase with leaked polyamines. However, the direct inhibition of polyamine oxidation was highly conceived to be the main cause for it. Based on the available information, including our present findings, we speculate that ROI in phase I, which is the prerequisite to the next phase II oxidative burst, is produced by NADPH oxidase immediately after elicitation, whereas ROI in phase II is produced mainly by polyamine degradation. We further speculate that oxidative burst in phase I may occur mainly in elicited cells and serve as one of the triggers for subsequent HR events, including phase II oxidative burst.

Signaling Pathways

MAPKs have long been considered to play a critical role in defense signal transduction and the defense gene activation (Tena et al., 2001; Zhang and Klessig, 2001; Jonak et al., 2002; MAPK Group, 2002). For example, SIPK and WIPK are well-known tobacco MAPKs that are activated together during defense responses against TMV infection, elicitors, and wounding (Zhang and Klessig, 1998; Romeis et al., 1999; Liu et al., 2003). However, the immediate triggers for activation of WIPK and SIPK themselves remain to be determined, although SIPK has been shown to respond to SA (Zhang and Klessig, 1997) and to ROI elements including hydrogen peroxide, superoxide, and ozone (Samuel et al., 2000). Activation of SIPK occurs within minutes, suggesting that the responsible stimulant is ROI rather than SA. This speculation is consistent with a report that the level of SIPK activity induced by cryptogein and harpin, a bacterial elicitor from Pseudomonas syringae pv syringae, did not change in NahG tobacco plants in which SA does not accumulate due to conversion into catechol (Lebrun-Garcia et al., 2002; Samuel et al., 2005). Our present finding that SIPK activity was drastically suppressed in BY2 cells, in which hydrogen peroxide production was suppressed, further supports this idea. In wild-type BY2 cells, SIPK was activated within 30 min after elicitation, when hydrogen peroxide production in phase II had not yet taken place. This implies that the activation of SIPK precedes polyamine oxidation, possibly being mediated by ROI generated in phase I through NADPH oxidase. However, activation of SIPK in phase I alone was not sufficient to induce programmed cell death, hydrogen peroxide in phase II appeared to be a prerequisite. This is in accordance with the finding that the phase I response does not necessarily correlate with disease resistance (Baker and Orlandi, 1995).

Overall, this study with transgenic lines and pharmacological experiments provided strong evidence that the majority of ROI in the late phase of oxidative burst is derived from polyamine oxidation, and that such ROI directly induces programmed cell death by activating the MAPK cascade.

MATERIALS AND METHODS

Plant Materials and Chemical Treatments

Tobacco (Nicotiana tabacum) cultured cells and BY2 cells were grown in Murashige and Skoog medium on a rotary shaker (115 rpm, 25°C) in the dark. Cells in stationary phase at day 7 after subculture were used after dilution to a 1:50 ratio in fresh medium. A 5-mL aliquot of cells at day 7 was added to 50 mL of fresh medium containing aphidicolin (10 μg mL−1) and cultured for 24 h for synchronization. For chemical treatments, synchronized cells were collected by filtration, washed with fresh medium, diluted into 50 mL fresh medium, and cultured for 1 h for acclimation. Cryptogein (Ponchet et al., 1999) was added at an appropriate concentration as indicated for each experiment. DFMO and guazatine were introduced into the medium 30 min before cryptogein treatment at the concentrations indicated. Polyamines or hydrogen peroxide were also added to the medium at the concentrations indicated in other groups. For polyamine oxidase assays, guazatine and FAD were added at appropriate concentrations as indicated in each experiment. Purified guazatine triacetate (Mr 535.73) was commercially obtained (Wako) and used at the concentration of 0.37 μm (0.2 μg mL−1).

DNA Laddering and Fragmentation

DNA was extracted by the cetyltrimethyl ammonium bromide method (Murray and Thompson, 1980), fractionated on agarose gel, and stained with ethidium bromide. DNA fragmentation was examined using the TUNEL method (in situ apoptosis detection kit, Takara; Tornusciolo et al., 1995) according to the manufacturer's protocol.

Quantitation of Cell Death

A 300 μL-aliquot of BY2 cells was incubated with 0.05% Evans blue for 15 min, washed several times to remove excess unbound dye, transferred into 70 μL fresh medium, and examined for dead cells by light microscopy. Data given are means from three replicate experiments, each with counts of approximately 300 cells.

Chemiluminescence Assay

Production of hydrogen peroxide and superoxide was determined by chemiluminescence using luminol and 2-methyl-6-phenyl-3, 7-dihydroimidazo-[1,2-α]pyrazin-3-one as the respective reaction reagents. BY2 cells were collected by filtration and washed several times with 5 mm MES buffer (pH 5.6) containing 175 mm mannitol, 0.5 mm CaCl2, and 0.5 mm K2SO4 (assay buffer). After resuspension in assay buffer at the concentration of 50 mg fresh weight per mL, samples were equilibrated by shaking at 115 rpm for 1 h at 25°C in the dark before cryptogein treatment. DFMO was added to the medium 30 min before cryptogein treatment. After the indicated time period, 25-μL aliquots of cell suspension from each sample were subjected to chemiluminescence assay. Measurement was performed as previously described (Yoda et al., 2003). Monitoring of hydrogen peroxide and potassium superoxide was performed with reference to results of adding known amounts of the two products.

Quantitation of Polyamines

BY2 cells were collected by filtration and washed several times with medium without Suc and 2,4-didhlorophenoxy-acetic acid. Proliferation was monitored by directly measuring fresh weights of cells. Polyamines from apoplasts were extracted with 50 mm MgCl2 (2.0 mL) in vacuo for 10 min. A 500-μL aliquot of medium supernatant was collected by centrifugation and the contained polyamines were derivatized with benzoyl chloride and separated by HPLC as described (Yoda et al., 2003). Standard curves for estimation were obtained by measuring a series of known amounts of each polyamine.

Assay for Polyamine Oxidase

BY2 cells were filtered through nylon mesh (20 μm) after an appropriate time period and washed with fresh medium. Apoplastic proteins were recovered by soaking cells in 10 mm Tris-HCl (pH 8.0; 2.5 mL) for 5 min and further in vacuo for 10 min. A 10-μl aliquot of recovered fluid was subjected to reaction with 5 mm of an appropriate polyamine substrate for 1 min, and hydrogen peroxide was measured as described (Yoda et al., 2003).

Scavenging Activity Assay

BY2 cells were synchronized and cultured in fresh medium for 1 h, when cryptogein was added to a final concentration of 50 nm. After incubation for an appropriate time period, cells were collected and resuspended at 50 mg fresh weight per mL of fresh medium containing 10 mm hydrogen peroxide. Scavenging activity was estimated by measuring decrease of relative light units (RLUs) in a chemiluminescence assay as described above after incubation at room temperature for the indicated time period.

Isolation of a cDNA for Polyamine Oxidase

Total RNA was prepared from TMV-infected tobacco leaves as described above and poly (A)+ RNA was purified with a mRNA purification kit (Pharmacia). A cDNA library was constructed using a λ-ZAP cDNA synthesis kit (Stratagene) with modification. The cDNAs, synthesized by reverse transcriptase (Super Script II, Gibco BRL) from 5 μg of poly (A)+ RNA with linker primers (anchored primers), were blunted and ligated to an EcoRI adaptor and after phosphorylation of adaptor ends, digestion with XhoI was performed, followed by fractionation by size with a SizeSep 400 Spun Column (Pharmacia). The fractionated cDNA was ligated to Uni-ZAP XR Vector Arms and packaged in λphage with MaxPlaxTM Lambda Packaging extract (Epicentre Technology). The resulting fragment, designated as NtPAO, was used as a probe to screen 2 × 105 plaques of the TMV-induced tobacco cDNA library. Positive plaques were purified through three successive plaque hybridizations and cDNA inserts were rescued in pBluescript SK(+) plasmids by ExAssist helper phage-mediated in vivo excision and sequenced.

Bacterial Expression and Activity Assay

The cDNA of NtPAO was amplified by PCR with a set of synthetic oligonucleotide primers containing a BamHI site (5′-GGATCCAAGGTGTTATCGGAGAATGG-3′) and a SalI site (5′-GTCGACTCATAAGATAGCCTCTGGAAGTC-3′), respectively, using cloned pBluscript II SK(-)-NtPAO as the template. The resulting fragment was ligated to the corresponding site of pGEX-4T vector, which was transformed into Escherichia coli BL21. Bacterial cells were grown at 37°C for 3 h and further incubated at 18°C for 12 h after addition of isopropyl β-d-thiogalactopyranoside to a final concentration of 0.5 mm. Recombinant protein was purified with glutathione-Sepharose 4B (Amersham) and subjected to polyamine oxidase assay as described above. Approximately 4 μg recombinant protein was used for one reaction.

Transient Expression and Subcellular Localization

A fragment of NtPAO was amplified by PCR with a set of synthetic oligonucleotide primers containing a SalI site (5′-GTCGACATGGCAACTCCCCG-3′) and a NcoI site (5′-CCATGGATAAGATAGCCTCTGGAAGTCC-3′), using cloned pBluscript II SK(-)-NtPAO as a template. This fragment was introduced into the SalI/NcoI site of the CaMV35S-sGFP(S65T)-NOS3′ vector, creating an in-frame fusion product between the coding region and GFP. The expression construct was delivered into onion (Allium cepa) epidermal cells by particle bombardment as previously described (Yoda et al., 2002) and after incubation of transformed cells for 8 h at 25°C in the dark, GFP was detected by fluorescence microscopy.

Immunoblot Analysis

Apoplastic fluids were recovered as described above. Cytoplasmic contents from remaining cells were extracted in 10 mm Tris-HCl (pH 6.8) with ultrasonic. For immunoblot analysis, 5 μg each of soluble protein from apoplasts and cytoplasm was separated on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidine fluoride membrane (Millipore). After blocking overnight in 137 mm NaCl, 2.7 mm KCl, 1.5 mm KH2PO4, and 8.1 mm Na2hPO4 (PBS) containing 5% skim milk at 4°C, the membrane was incubated with antibodies against PEPC diluted with PBS for 1 h at room temperature. After several washings with PBS containing 0.1% Tween 20, the membrane was incubated with horseradish peroxidase-conjugated anti-goat IgG antibodies (Bio-Rad). Antibody-antigen complex was detected using ECL system (Amersham).

Transformation of BY2 Cells

To generate transgenic BY2 cells with suppressed polyamine oxidase activity, the pKANNIBAL (Wesley et al., 2001) vector designed for producing hairpin RNA with a loop was employed. For sense orientation, the coding region of NtPAO cDNA was amplified by PCR with a set of synthetic oligonucleotide primers containing a XhoI site (5′-AGCTCGAGGTAATCGGTTTCAGC-3′) and a KpnI site (5′-GTGGTACCACTAAGGAAGAGTTGTCG-3′). For antisense orientation, the same region was amplified with a set of synthetic oligonucleotide primers containing a BamHI site (5′-ACGGATCCGGAATAATCGGTTTCAGC-3′) and a ClaI site (5′-GTATCGATACTAAGGAAGAGTTGTCG-3′). The obtained fragments were introduced into the pGEM-T Easy vector following digestion with designed restriction enzymes. Digested fragments were ligated to the corresponding sites of the pKANNIBAL vector and the resulting plasmid was digested with NotI and ligated to the corresponding pART27 vector, which was introduced into Agrobacterium tumefaciens strain EHA105 cells. The final construct was introduced into tobacco BY2 cells as previously described (Yamaguchi et al., 2003).

RNA Isolation and Gel-Blot Analysis

Total RNA was isolated by the acid guanidine thiocyanate-phenol/chloroform method (Chomczynski and Sacchi, 1987) and gel-blot analyses were performed as detailed earlier (Yoda et al., 2002) with probes produced as previously described (Yoda et al., 2003). Probes for HSR203J and HIN1were synthesized with a pair of specific primers: forward (5′-ATGGTTCATGAAAAGCAAGTGATAGAGG-3′) and reverse (5′-GCTTGTTGATGAACTCTGCAACGGCTTC-3′) for HSR203J, and forward (5′-CCCTTCCATTCCGCCACCAGCAAAATCC-3′) and reverse (5′-CTACCAATCAAGATGGCATCTGGTTTCC-3′) for HIN1.

In-Gel Kinase Activity Assay

In-gel kinase assays were performed as described previously (Zhang and Klessig, 1997). Extracts containing 20 μg of protein were subjected to electrophoresis on 10% SDS-polyacrylamide gels embedded with 0.25 mg/mL of myelin basic protein as a substrate for kinase. After electrophoresis, SDS was removed by washing the gels with washing buffer (25 mm Tris-HCl, pH 7.5, 0.5 mm dithiothreitol [DTT], 0.1 mm Na3V04, 5 mm NaF, 0.5 mg mL−1 bovine serum albumin, and 0.1% Triton X-100 [v/v]) three times, each for 30 min at room temperature. Protein kinase was allowed to renature in 25 mm Tris-HCl, pH 7.5, 1 mm DTT, 0.1 mm Na3V04, and 5 mm NaF at 4°C for 16 h with three changes of the buffer, and the gels were then incubated at room temperature in a 30 mL reaction buffer (25 Tris-HCl, pH 7.5, 2 mm EGTA, 12 mm MgCl2, 1 mm DTT, and 0.1 mm Na3V04) with 200 nm ATP plus 50 μCi [γ-32P]ATP (3,000 Ci/mmol) for 60 min. The reaction was stopped by transferring the gel into 5% trichloroacetic acid (w/v)/1% NaPPi (w/v). The unincorporated [γ-32P]ATP was removed by washing in the same solution for at least 6 h with five changes and gels were then finally dried on Whatman 3MM paper for exposure of x-ray film.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AB200262.

Acknowledgments

The authors thank Drs. Michel Ponchet and Jean-Claude Pernollet (Institut National de la Recherche Agronomique, France), and Toyoki Amano (Shizuoka University) for generous provision of cryptogein; Takashi Hashimoto (Nara Institute of Science and Technology) and Kenzo Nakamura (Nagoya University) for cDNA clones of spermidine synthase and S-adenosyl-methionine decarboxylase, respectively; and Dr. Malcolm Moore (Intermal, Nagoya) for critical reading of the manuscript.

Notes

1This work was supported by a grant from the Research for the Future Program of the Japan Society for the Promotion of Science and by a Grant-in-Aid for the 21st Century Center of Excellence Research from the Ministry of Education, Culture, Sports, Science and Technology.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hiroshi Yoda (pj.tsian.sb@adoy-h).

www.plantphysiol.org/cgi/doi/10.1104/pp.106.080515

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