Polyamine oxidase is one of the key elements for oxidative burst to induce programmed cell death in tobacco cultured cells

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 alpha-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.     

Functions of amine oxidases in plant development and defence

Copper amine oxidases and flavin-containing amine oxidases catalyse the oxidative de-amination of polyamines, which are ubiquitous compounds essential for cell growth and proliferation. Far from being only a means of degrading cellular polyamines and, thus, contributing to polyamine homeostasis, amine oxidases participate in important physiological processes through their reaction products. In plants, the production of hydrogen peroxide (H(2)O(2)) deriving from polyamine oxidation has been correlated with cell wall maturation and lignification during development as well as with wound-healing and cell wall reinforcement during pathogen invasion. As a signal molecule, H(2)O(2) derived from polyamine oxidation mediates cell death, the hypersensitive response and the expression of defence genes. Furthermore, aminoaldehydes and 1,3-diaminopropane from polyamine oxidation are involved in secondary metabolite synthesis and abiotic stress tolerance.     

Short-term polyamine response in TMV-inoculated hypersensitive and susceptible tobacco plants

The short-term polyamine response to inoculation, with tobacco mosaic virus (TMV), of TMV-inoculated NN (hypersensitive) and nn (susceptible) plants of Nicotiana tabacum (L.) cv. Samsun was investigated. Free and conjugated polyamine concentrations, putrescine biosynthesis, evaluated through arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activities, and putrescine oxidation, via diamine oxidase (DAO) activity, were analysed during the first 24 h from inoculation. Results were compared with those of mock-inoculated control plants. In NN TMV-inoculated plants undergoing the hypersensitive response (HR), free putrescine and spermidine concentrations had increased after 5 h compared with controls; polyamine conjugates also tended to increase compared with controls. In both virus- and mock-inoculated plants, ADC and ODC activities generally increased whereas DAO activity, which was present in controls, was detectable only in traces in inoculated tissues.
In TMV-infected susceptible plants, free putrescine and spermidine concentrations were lower at 5 h relative to controls, as were polyamine conjugates. No differences were revealed in ADC and ODC activities whereas DAO activity was not detectable. These results further support the hypothesis that polyamines are involved in the response of tobacco to TMV and that, only a few hours after inoculation, the response of hypersensitive plants is distinct from that of susceptible ones.     

Identification, characterization, and purification of a tobacco endonuclease activity induced upon hypersensitive response cell death.

Programmed cell death (pcd) is activated during the hypersensitive response (HR) of plants to avirulent pathogens. We have recently shown that, similar to pcd in animal cells, nuclei of cells undergoing HR cell death contain fragmented nuclear DNA (nDNA). Here, we report that cell death occurring during the HR is accompanied by an increase in the activity of several deoxyribonucleases. Induction of nuclease activities was coordinated with cell death and may account for the degradation of nDNA during the HR. HR-associated nuclease activities were not induced during senescence, following necrotic cell death resulting from abiotic stress, or in response to induction of plant defense mechanisms by salicylic acid. HR-associated nuclease activities were stimulated by Ca2+ and inhibited by EGTA, EDTA, and Zn2+. At least one of the HR-associated nuclease activities was detected in nuclei purified from leaves undergoing pcd. A nuclease with an electrophoretic mobility similar to that of the nuclease activity found in nuclei isolated from leaves undergoing HR cell death was purified. Our findings are in accordance with some of the biochemical events that occur during pcd in animal cells. However, further analysis of the pattern of nDNA fragmentation and the corresponding structural changes that occur in the nuclei of tobacco cells undergoing HR cell death revealed that these features may have differences from those that take place during apoptosis in animal cells.     

Expression of the baculovirus p35 protein in tobacco affects cell death progression and compromises N gene-mediated disease resistance response to Tobacco mosaic virus

The p35 protein from baculovirus is a broad-range caspase inhibitor and suppresses programmed cell death in animals. We report here the effects of transgenic expression in tobacco of the p35 protein during the hypersensitive response (HR). Expression of p35 causes partial inhibition of nonhost HR triggered by bacteria and gene-for-gene HR triggered by virus. Infection of p35-expressing tobacco plants with Tobacco mosaic virus (TMV) disrupts N-mediated disease resistance, causing systemic spreading of the virus within a resistant background. Mutant variants altered in aspartate residues within the loop region of p35 are inefficient substrates for caspases in vitro, and they do not suppress caspase proteolytic activity in animal systems. Tobacco plants expressing these mutant variants of the p35 protein do not show inhibition of HR cell death or enhanced virus systemic movement. Thus, HR inhibition and TMV systemic spreading phenotype in p35-expressing plants correlate with the ability of the p35 protein to suppress caspase activity in animal systems. In addition, a C-terminal truncated variant of p35 is unable to suppress cell death in animals as well as HR cell death in transgenic tobacco. Our results provide evidence for the participation of caspase-like proteases during the HR. In addition, they suggest that timely activation of cell death is necessary for effective TMV containment within the primary infection site.     

Identification of Tobacco HIN1 and Two Closely Related Genes as Spermine-Responsive Genes and their Differential Expression During the Tobacco Mosaic Virus-Induced Hypersensitive Response and During Leaf- and Flower-Senescence

Previously we showed that the polyamine spermine (Spm) specifically leads to mitochondrial dysfunction in tobacco that is followed by the activation of salicylic acid-induced protein kinase and wound-induced protein kinase. To identify the possible downstream components of the Spm signalling pathway, we isolated Spm-responsive genes by a differential hybridization approach. This showed that the harpin-induced 1 (HIN1) gene is responsive to Spm. Genomic Southern analysis showed that HIN1 constitutes a multi-gene family and this led to the isolation of two novel HIN1 -like tobacco cDNAs that we designated as HIN9 and HIN18. Both genes are also responsive to Spm, albeit HIN18 is induced weakly compared to HIN1 and HIN9. As HIN1 is up-regulated both during the hypersensitive response (HR) generated by an incompatible plant-pathogen interaction and during senescence, we compared the expression of the three HIN1 family genes in these situations. All three were responsive to HR due to Tobacco mosaic virus infection, although HIN18 was less efficiently induced, and HIN1 and HIN18 were both strongly up-regulated during leaf- and flower-senescence. This suggests that the signalling pathways in the HR and senescence overlap somehow but are distinct. That HIN1 and its closely related genes are Spm-responsive genes also supports the idea that Spm plays a role as a signal transmitter in the HR process.     

Inhibition of Programmed Cell Death in Tobacco Plants during a Pathogen-Induced Hypersensitive Response at Low Oxygen Pressure

The hypersensitive response (HR) of plants to invading pathogens is thought to involve a coordinated activation of plant defense mechanisms and programmed cell death (pcd). To date, little is known about the mechanism underlying death of plant cells during this response. In addition, it is not known whether suppression of pcd affects the induction of other defense mechanisms during the HR. Here, we report that death of tobacco cells (genotype NN) infected with tobacco mosaic virus (TMV) is inhibited at low oxygen pressure. In contrast, virus replication and activation of defense mechanisms, as measured by synthesis of the pathogenesis-related protein PR-1a, were not inhibited at low oxygen pressure. Bacterium-induced pcd was also inhibited at low oxygen pressure. However, pcd induced by TMV or bacteria was not inhibited in transgenic tobacco plants expressing the mammalian anti-pcd protein Bcl-XL. Our results suggest that ambient oxygen levels are required for efficient pcd induction during the HR of plants and that activation of defense responses can be uncoupled from cell death. Furthermore, pcd that occurs during the interaction of tobacco with TMV or bacteria may be distinct from some cases of pcd or apoptosis in animals that are insensitive to low oxygen or inhibited by the Bcl-XL protein.     

Polyamine depletion induces apoptosis through mitochondria-mediated pathway

Polyamines, namely putrescine, spermidine, and spermine, are essential for cell survival and proliferation. A decrease in intracellular polyamine levels is associated with apoptosis. In this study, we used inhibitors of polyamine biosynthesis to examine the effect of polyamine depletion. A combination of inhibitors of ornithine decarboxylase, S-adenosylmethionine decarboxylase, or spermidine synthase decreased intracellular polyamine levels and induced cell death in a WEHI231 murine B cell line. These cells exhibited apoptotic features including chromatin condensation and oligonucleosomal DNA fragmentation. Addition of exogenous polyamines reversed the observed features of apoptotic cell death. Similar effects were also observed in other cell lines: a human B cell line Ramos and a human T cell line Jurkat. Depletion of polyamines induced activation of caspase-3 and disruption of the mitochondrial membrane potential (Delta psi m). Inhibition of caspase activities by an inhibitor prevented the apoptotic nuclear changes but not Delta psi m disruption induced by polyamine depletion. Overexpression of Bcl-xl, an anti-apoptotic Bcl-2 family protein, completely inhibited Delta psi m disruption, caspase activation, and cell death. These results indicate that the depletion of intracellular polyamines triggers the mitochondria-mediated pathway for apoptosis, resulting in caspase activation and apoptotic cell death.     

Effect of the polyamine analogue N1,N11-diethylnorspermine on cell survival and susceptibility to apoptosis of human chondrocytes

Chondrocyte survival is closely linked to cartilage integrity, and forms of chondrocyte apoptotic death can contribute to cartilage degeneration in articular diseases. Since growing evidence also implicates polyamines in the control of cell death, we have been investigating the role of polyamine metabolism in chondrocyte survival and apoptosis. Treatment of human C-28/I2 chondrocytes with N(1),N(11)-diethylnorspermine (DENSPM), a polyamine analogue with clinical relevance as an experimental anticancer agent, inhibited polyamine biosynthesis and induced polyamine catabolism, thus rapidly depleting all main polyamines. DENSPM did not increase significantly caspase activity, but provoked a late cell death associated to DNA fragmentation. A short treatment with DENSPM did not reduce cell viability when given alone, but enhanced caspase-3 and -9 activation in chondrocytes exposed to tumor necrosis factor-alpha (TNF) and cycloheximide (CHX). A longer treatment with DENSPM however reduced caspase response to TNF plus CHX. Depletion of all polyamines obtained by specific inhibitors of polyamine biosynthesis did not cause cell death and contrasted apoptosis by decreasing caspase activities. In conclusion, following DENSPM treatment, C-28/I2 chondrocytes are initially sensitized to caspase 9-dependent apoptosis in the presence of TNF and CHX and may eventually undergo a late and mainly caspase-independent cell death in the absence of other stimuli. Moreover, these results indicate that a reduction of polyamine levels not only leads to inhibition of cell proliferation, but also of caspase-mediated pathways of chondrocyte apoptosis.     

Involvement of the small GTPase Rac in the defense responses of tobacco to pathogens

During the hypersensitive response (HR), plants accumulate reactive oxygen species (ROS) that are likely generated at least in part by an NADPH oxidase similar to that found in mammalian neutrophils. An essential regulator of mammalian NADPH oxidase is the small GTP-binding protein Rac. To investigate whether Rac also regulates the pathogen-induced oxidative burst in plants, a dominant negative form of the rice OsRac1 gene was overexpressed in tobacco carrying the N resistance gene. Following infection with Tobacco mosaic virus (TMV), DN-OsRacl plants developed smaller lesions than wild-type plants, accumulated lower levels of lipid peroxidation products, and failed to activate expression of antioxidant genes. These results, combined with the demonstration that superoxide and hydrogen peroxide levels were reduced in DN-OsRacl tobacco developing a synchronous HR triggered by transient expression of the TMV p50 helicase domain or the Pto and AvrPto proteins, suggest that ROS production is impaired. The dominant negative effect of DN-OsRacl could be rescued by transiently overexpressing the wild-type OsRac1 protein. TMV-induced salicylic acid accumulation also was compromised in DN-OsRacl tobacco. Interestingly, while systemic acquired resistance to TMV was not impaired, nonhost resistance to Pseudomonas syringae pv. maculicola ES4326 was suppressed. Thus, the effect DN-OsRac1 expression exerts on the resistance signaling pathway appears to vary depending on the identity of the inoculated pathogen.     

Tobamovirus coat protein CPCg induces an HR-like response in sensitive tobacco plants

When inoculated into sensitive tobacco Xanthi-nn plants, the crucifer and garlic-infecting Tobacco mosaic virus (TMV-Cg) induces local necrotic lesions that resemble those seen in the hypersensitive response (HR) of resistant tobacco plants. However, unlike these, tobacco Xanthi-nn plants do not become resistant to infection and the virus spreads systemically causing a severe disease characterized by necrotic lesions throughout the plant. To identify the viral protein that elicits this necrotic response, we used a set of hybrid viruses constructed by combination of TMV-Cg and the tobacco mosaic virus strain U1 (TMV-U1). In this study we present evidence that the coat protein of TMV-Cg (CPCg) is the elicitor of the necrotic response in tobacco Xanthi-nn plants. Local and systemic necrotic lesions induced by TMV-Cg and by the hybrid U1-CPCg -that carries CPCg in a TMV-U1 context- are characterized by cell death and by the presence of autoflorescent phenolic compounds and H2O2, just like the HR lesions. In addition, defense-related genes and detoxifying genes are induced in tobacco Xanthi-nn plants after TMV-Cg and U1-CPCg inoculation. We postulate that in our system, CPCg is recognized by sensitive tobacco plants that mount an incomplete defense response. We call this an HR-like since it is not enough to induce plant resistance.     

Role of Polyamines in Apoptosis and Other Recent Advances in Plant Polyamines

Numerous citations in the literature indicate that polyamines are intensively studied in plants. Polyamines are implicated in many functions of the plant cell. Excellent recent reviews cover much of this original research. This article is focused primarily on literature that relates research on the role of polyamines in apoptosis and programmed cell death in both plants and animals. Apoptosis and programmed cell death are considerably better studied in animal systems, and this review demonstrates that the role of polyamines in these processes in plant systems are remarkably congruent with what is known in animal systems. In addition, key recent research reports are reviewed that describe the functional analysis of key polyamine biosynthesis genes in plants in relation to responses to environmental stress signals. Molecular analysis is providing strong evidence for the polyamine biosynthetic pathways to play major roles in ameliorating plant responses to abiotic stresses.     

Killer polyamines?

Mammalian cells can rapidly make large changes in their rate of polyamine biosynthesis in response to mitogenic and trophic signals. However, cultured cells seem to grow adequately as long as they are supplied a steady but unregulated supply of polyamines. This implies that complex and rapid changes in polyamine synthesis serve a function in a special rather than a general biological context. We suggest that the appropriate context in which regulation of polyamines mediates crucial functions is the mammalian embryo and that one function of polyamines is to act as substrate in an oxidative pathway that arbitrates programmed cell death.     

Involvement of Hydrogen Peroxide Generated by Polyamine Oxidative Degradation in the Development of Lateral Roots in Soybean

In order to determine whether hydrogen peroxide （H2O2） generated by polyamlne oxidative degradation Is Involved In the development of lateral roots In soybean, the length and the number of lateral roots, the actlvltlea of polyamlne oxldases and dlamlne oxldases, and the endogenous free polyamlne and H2O2 content were analyzed In soybean （Giycine max （Linn.） Merr.） main roots of 2-d-old seedlings after treatments for 2 d with exogenous β-hydroxyethylhydrazine （an Inhibitor of polyamlne oxldases）, H202, putresclne, cyclohexylamlne （an Inhibitor of spermidine synthase） or N,N＇-dimethylthlourea （a scavenger of hydrogen peroxide）.β-hydroxyethylhydrazlne treatment strongly Inhibited the development of lateral roots In soybean seedlings, reduced the activities of polyamine oxldases and dlamlne oxidases, decreased H2O2 levels, and led to the accumulation of endogenous polyamlnes In the main roots. The inhibitory effect of β-hydroxyethylhydrazlne on root development could be alleviated by exogenously applied 10 μmol/L H2O2 （a major product of polyamlne oxidation）. Treatment with cyclohexylamlne and putresclne promoted root growth slightly, but treatment with cyclohexylamlne plus N,N＇dlmethylthlourea or putresclne plus N,N＇-dlmethylthlourea prevented the development of soybean lateral roots. The effects of these treatments on the development of soybean lateral roots were consistent with the changes In endogenous H2O2 levels. These results suggest that the development of soybean lateral roots Is associated with the oxidative degradation of polyamlnes, and that their products, especially H2O2, are likely to play an Important role In the growth of soybean lateral roots.     

The helicase domain of the TMV replicase proteins induces the N-mediated defence response in tobacco

Tobacco mosaic virus (TMV) induces the hypersensitive response (HR) in tobacco plants containing the N gene. This defence response is characterized by cell death at the site of virus infection and inhibition of viral replication and movement. A previous study indicated that a portion of the TMV replicase containing a putative helicase domain is involved in HR induction. Here, this observation is confirmed and extended by showing that non-viral expression of a 50 kDa TMV helicase fragment (p50) is sufficient to induce the N-mediated HR in tobacco. Like the HR elicited by TMV infection, transgenic expression of p50 induces a temperature-sensitive defence response. We demonstrate that recombinant p50 protein has ATPase activity, as suggested by the presence of conserved sequence motifs found in ATPase/helicase enzymes. A point mutation that alters one of these motifs abolishes ATPase activity in vitro but does not affect HR induction. These results suggest that features of the TMV helicase domain, independent of its enzymatic activity, are recognized by N-containing tobacco to induce TMV resistance.     

Polyamines and apoptosis

The natural polyamines putrescine, spermidine and spermine are in multiple ways involved in cell growth and the maintenance of cell viability. In the course of the last 15 years more and more evidence hinted also at roles in gene regulation. It is therefore not surprising that the polyamines are involved in events inherent to genetically programmed cell death. Following inhibition of ornithine decarboxylase, a key step in polyamine biosynthesis, numerous links have been identified between the polyamines and apoptotic pathways. Examples of activation and prevention of apoptosis due to polyamine depletion are known for several cell lines. Elevation of polyamine concentrations may lead to apoptosis or to malignant transformation. These observations are discussed in the present review, together with possible mechanisms of action of the polyamines. Contradictory results and incomplete information blur the picture and complicate interpretation. Since, however, much interest is focussed at present on all aspects of programmed cell death, a considerable progress in the elucidation of polyamine functions in apoptotic signalling pathways is expected, even though enormous difficulties oppose pinpointing specific interactions of the polyamines with pro- and anti-apoptotic factors. Such situation is quite common in polyamine research.