Spermidine exodus and oxidation in the apoplast induced by abiotic stress is responsible for H2O2 signatures that direct tolerance responses in tobacco

Polyamines (PAs) exert a protective effect against stress challenges, but their molecular role in this remains speculative. In order to detect the signaling role of apoplastic PA-derived hydrogen peroxide (H2O2) under abiotic stress, we developed a series of tobacco (Nicotiana tabacum cv Xanthi) transgenic plants overexpressing or downregulating apoplastic polyamine oxidase (PAO; S-pao and A-pao plants, respectively) or downregulating S-adenosyl-l-methionine decarboxylase (samdc plants). Upon salt stress, plants secreted spermidine (Spd) into the apoplast, where it was oxidized by the apoplastic PAO, generating H2O2. A-pao plants accumulated less H2O2 and exhibited less programmed cell death (PCD) than did wild-type plants, in contrast with S-pao and samdc downregulating plants. Induction of either stress-responsive genes or PCD was dependent on the level of Spd-derived apoplastic H2O2. Thus, in wild-type and A-pao plants, stress-responsive genes were efficiently induced, although in the latter at a lower rate, while S-pao plants, with higher H2O2 levels, failed to accumulate stress-responsive mRNAs, inducing PCD instead. Furthermore, decreasing intracellular PAs, while keeping normal apoplastic Spd oxidation, as in samdc downregulating transgenic plants, caused enhanced salinity-induced PCD. These results reveal that salinity induces the exodus of Spd into the apoplast, where it is catabolized by PAO, producing H2O2. The accumulated H2O2 results in the induction of either tolerance responses or PCD, depending also on the levels of intracellular PAs.     

Involvement of polyamine oxidase in wound healing

Hydrogen peroxide (H(2)O(2)) is involved in plant defense responses that follow mechanical damage, such as those that occur during herbivore or insect attacks, as well as pathogen attack. H(2)O(2) accumulation is induced during wound healing processes as well as by treatment with the wound signal jasmonic acid. Plant polyamine oxidases (PAOs) are H(2)O(2) producing enzymes supposedly involved in cell wall differentiation processes and defense responses. Maize (Zea mays) PAO (ZmPAO) is a developmentally regulated flavoprotein abundant in primary and secondary cell walls of several tissues. In this study, we investigated the effect of wounding on ZmPAO gene expression in the outer tissues of the maize mesocotyl and provide evidence that ZmPAO enzyme activity, protein, and mRNA levels increased in response to wounding as well as jasmonic acid treatment. Histochemically detected ZmPAO activity especially intensified in the epidermis and in the wound periderm, suggesting a tissue-specific involvement of ZmPAO in wound healing. The role played by ZmPAO-derived H(2)O(2) production in peroxidase-mediated wall stiffening events was further investigated by exploiting the in vivo use of N-prenylagmatine (G3), a selective and powerful ZmPAO inhibitor, representing a reliable diagnostic tool in discriminating ZmPAO-mediated H(2)O(2) production from that generated by peroxidase, oxalate oxidase, or by NADPH oxidase activity. Here, we demonstrate that G3 inhibits wound-induced H(2)O(2) production and strongly reduces lignin and suberin polyphenolic domain deposition along the wound, while it is ineffective in inhibiting the deposition of suberin aliphatic domain. Moreover, ZmPAO ectopic expression in the cell wall of transgenic tobacco (Nicotiana tabacum) plants strongly enhanced lignosuberization along the wound periderm, providing evidence for a causal relationship between PAO and peroxidase-mediated events during wound healing.     

Heterologous expression and biochemical characterization of a polyamine oxidase from Arabidopsis involved in polyamine back conversion

Polyamine oxidase (PAO) is a flavin adenine dinucleotide-dependent enzyme involved in polyamine catabolism. Animal PAOs oxidize spermine (Spm), spermidine (Spd), and/or their acetyl derivatives to produce H2O2, an aminoaldehyde, and Spd or putrescine, respectively, thus being involved in a polyamine back-conversion pathway. On the contrary, plant PAOs that have been characterized to date oxidize Spm and Spd to produce 1,3-diaminopropane, H2O2, and an aminoaldehyde and are therefore involved in the terminal catabolism of polyamines. A database search within the Arabidopsis (Arabidopsis thaliana) genome sequence showed the presence of a gene (AtPAO1) encoding for a putative PAO with 45% amino acid sequence identity with maize (Zea mays) PAO. The AtPAO1 cDNA was isolated and cloned in a vector for heterologous expression in Escherichia coli. The recombinant protein was purified by affinity chromatography on guazatine-Sepharose 4B and was shown to be a flavoprotein able to oxidize Spm, norspermine, and N1-acetylspermine with a pH optimum at 8.0. Analysis of the reaction products showed that AtPAO1 produces Spd from Spm and norspermidine from norspermine, demonstrating a substrate oxidation mode similar to that of animal PAOs. To our knowledge, AtPAO1 is the first plant PAO reported to be involved in a polyamine back-conversion pathway.     

Functional diversity inside the Arabidopsis polyamine oxidase gene family

Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. All so far characterized PAOs from monocotyledonous plants, such as the apoplastic maize PAO, oxidize spermine (Spm) and spermidine (Spd) to produce 1,3-diaminopropane, H(2)O(2), and an aminoaldehyde, and are thus considered to be involved in a terminal catabolic pathway. Mammalian PAOs oxidize Spm or Spd (and/or their acetyl derivatives) differently from monocotyledonous PAOs, producing Spd or putrescine, respectively, in addition to H(2)O(2) and an aminoaldehyde, and are therefore involved in a polyamine back-conversion pathway. In Arabidopsis thaliana, five PAOs (AtPAO1-AtPAO5) are present with cytosolic or peroxisomal localization and three of them (the peroxisomal AtPAO2, AtPAO3, and AtPAO4) form a distinct PAO subfamily. Here, a comparative study of the catalytic properties of recombinant AtPAO1, AtPAO2, AtPAO3, and AtPAO4 is presented, which shows that all four enzymes strongly resemble their mammalian counterparts, being able to oxidize the common polyamines Spd and/or Spm through a polyamine back-conversion pathway. The existence of this pathway in Arabidopsis plants is also evidenced in vivo. These enzymes are also able to oxidize the naturally occurring uncommon polyamines norspermine and thermospermine, the latter being involved in important plant developmental processes. Furthermore, data herein reveal some important differences in substrate specificity among the various AtPAOs, which suggest functional diversity inside the AtPAO gene family. These results represent a new starting point for further understanding of the physiological role(s) of the polyamine catabolic pathways in plants.     

Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H(2)O(2)-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H(2)O(2) only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H(2)O(2) produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.     

Transgenic tobacco plants overexpressing polyamine oxidase are not able to cope with oxidative burst generated by abiotic factors

The molecular and biochemical mechanism(s) of polyamine (PA) action remain largely unknown. Transgenic tobacco plants overexpressing polyamine oxidase (PAO) from Zea mays exhibited dramatically increased expression levels of Mpao and high 1,3-diaminopropane (Dap) content. All fractions of spermidine and spermine decreased significantly in the transgenic lines. Although Dap was concomitantly generated with H(2)O(2) by PAO, the latter was below the detection limits. To show the mode(s) of H(2)O(2) scavenging, the antioxidant machinery of the transgenics was examined. Specific isoforms of peroxidase, superoxide dismutase and catalase were induced in the transgenics but not in the wild-type (WT), along with increase in activities of additional enzymes contributing to redox homeostasis. One would expect that because the antioxidant machinery was activated, the transgenics would be able to cope with increased H(2)O(2) generated by abiotic stimuli. However, despite the enhanced antioxidant machinery, further increase in the intracellular reactive oxygen species (ROS) by exogenous H(2)O(2), or addition of methylviologen or menadione to transgenic leaf discs, resulted in oxidative stress as evidenced by the lower quantum yield of PSII, the higher ion leakage, lipid peroxidation and induction of programmed cell death (PCD). These detrimental effects of oxidative burst were as a result of the inability of transgenic cells to further respond as did the WT in which induction of antioxidant enzymes was evident soon following the treatments. Thus, although the higher levels of H(2)O(2) generated by overexpression of Mpao in the transgenics, with altered PA homeostasis, were successfully controlled by the concomitant activation of the antioxidant machinery, further increase in ROS was detrimental to cellular functions and induced the PCD syndrome.     

Flavin-containing polyamine oxidase is a hydrogen peroxide source in the oxidative response to the protein phosphatase inhibitor cantharidin in Zea mays L

In this study, the specific contribution of polyamine oxidase (PAO), a hydrogen peroxide (H2O2)-producing enzyme, to the oxidative burst induced in maize mesocotyl by the phosphatase inhibitor cantharidin was examined. For this purpose, a pharmacological approach was applied using, either in vitro or in vivo, two strong inhibitors of maize PAO (MPAO), N-prenylagmatine (G3) and its structural analogue Ro5, as well as diphenyleneiodonium (DPI), an inhibitor of the phagocyte NAD(P)H oxidase. DPI was shown to be a good MPAO inhibitor in vitro. G3, Ro5, and DPI were very effective in inhibiting in vivo the extracellular accumulation of H2O2 that is released by mesocotyl segments upon spermidine supply. G3 and Ro5 did not show any inhibition in vitro of either horseradish peroxidase or barley oxalate oxidase. Moreover, G3 and Ro5 did not inhibit the extracellular accumulation of superoxide radical that is released in vivo upon NADH supply. G3, Ro5, and DPI strongly affected H2O2 production induced in maize mesocotyl by cantharidin. Histochemical localization of H2O2 in cantharidin-treated mesocotyl cross-sections revealed an increase of H2O2-specific staining in the epidermal and subepidermal tissues. The effect was also inhibited by G3 and DPI. Moreover, an increase in MPAO activity was observed in the same tissues upon cantharidin treatment. All these data suggest that G3 and Ro5 behave as powerful and selective inhibitors of MPAO activity either in vitro or in vivo and that MPAO activity contributes to a major part of the cantharidin-induced H2O2 synthesis in the apoplastic milieu of maize mesocotyl.     

Polyamines promote root elongation and growth by increasing root cell division in regenerated Virginia pine (Pinus virginiana Mill.) plantlets

Polyamines have been demonstrated to play an important role in adventitious root formation and development in plants. Here, we present a detailed analysis of influence of exogenously added polyamines on adventitious root development and its relationship to cold tolerance in Virginia pine (Pinus virginia Mill.). Our results demonstrated that polyamines putrescine (Put), spermidine (Spd), and spermine (Spm) at 0.001 mM improve rooting frequency and promote root elongation. Put, Spd, and Spm at 0.01–1 mM decrease rooting frequency and reduce root elongation root elongation. Measurements of diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.4.3.4) activities showed that higher DAO and PAO enzyme activities were obtained when high concentrations of polyamines were applied and when plantlets were treated for 5–7 week at 4°C and 16°C. Survival rate of plantlets increased with the treatment of polyamines at low temperature. Polyamines increased mitotic index of cells in root tips of regenerated plantlet cultured on medium containing 0.001 μM Put, Spd, or Spm, but did not increase mitotic index in tissues of needle tips of the same plantlets. These results demonstrated that polyamines promote root elongation and growth by increasing root cell division in regenerated Virginia pine plantlets.     

干旱胁迫下多裂骆驼蓬(Peganum multisectum Bobr)叶片乙烯释放和多胺含量变化与活性氧积累的关系

通过盆栽土培试验研究了干旱胁迫下多裂骆驼蓬(Peganum multisectum Bobr)叶片乙烯释放、多胺含量、活性氧水平及细胞膜透性的动态变化.结果表明,随着土壤干旱强度的增加和胁迫时间的延长,多裂骆驼蓬叶片内丙二醛(MDA)含量和细胞膜透性呈递增变化趋势;轻度胁迫下,乙烯产生速率、多胺(Put、Spm、Spd)、活性氧(O2-·、H2O2)和MDA含量及多胺氧化酶(PAO)活性随胁迫时间延长无明显变化;中度和重度胁迫初期(胁迫后0～30d)腐胺(Put)、精胺(Spm)和亚精胺(Spd)含量快速提高,乙烯生成速率下降,而O2-·、H2O2、MDA含量和PAO活性相对平稳;胁迫后期(胁迫后45～75d)乙烯生成速率增高,Put、Spm和Spd含量下降, O2-·、H2O2和MDA含量大幅度增加,膜透性增大,且乙烯释放与O2-·、H2O2含量呈显著正相关.表明中度和重度胁迫下多裂骆驼蓬叶片存在乙烯和多胺合成的相互制约,乙烯释放增加诱导活性氧积累导致膜透性急剧增大.     

水分胁迫诱导玉米叶片质外体产生H2O2机理

通过组织化学染色、电镜观察、酶活性分析对水分胁迫诱导玉米叶片质外体产生H2O2进行了研究。结果表明:水分胁迫能够诱导玉米叶片内源ABA的积累,ABA参与了水分胁迫诱导的玉米叶片H2O2的产生,质膜NADPH氧化酶、细胞壁过氧化物酶(POD)以及质外体多胺氧化酶(PAO)是水分胁迫诱导玉米细胞在质外体产生H2O2的来源,其中质膜NADPH氧化酶是主要来源;内源ABA的积累参与了水分胁迫激活的质膜NADPH氧化酶、细胞壁POD和质外体PAO活性的提高。研究认为,水分胁迫诱导玉米细胞在质外体产生H2O2可能是由于水分胁迫下内源ABA的积累通过激活质膜NADPH氧化酶、细胞壁POD以及质外体PAO的活性而实现的。     

Hydrogen peroxide induces programmed cell death features in cultured tobacco BY-2 cells, in a dose-dependent manner

Active oxygen species (AOS), especially hydrogen peroxide, play a critical role in the defence of plants against invading pathogens and in the hypersensitive response (HR). This is characterized by the induction of a massive production of AOS and the rapid appearance of necrotic lesions is considered as a programmed cell death (PCD) process during which a limited number of cells die at the site of infection. This work was aimed at investigating the mode of cell death observed in cultures of BY-2 tobacco cells exposed to H(2)O(2). It was shown that H(2)O(2) is able to induce various morphological cell death features in cultured tobacco BY-2 cells. The hallmarks of cell death observed with fluorescent and electron microscopy differed greatly with the amount of H(2)O(2) added to the cell culture. The appearance of nuclear fragmentation similar to 'apoptotic bodies' associated with a fragmentation of the nuclear DNA into small fragments appear for almost 18% of the cells treated with 12.5 mM H(2)O(2). The early stages of the induction of this PCD process consisted in cell shrinkage and chromatin condensation at the periphery of the nucleus. Above 50 mM, H(2)O(2) induces high necrotic cell death. These data suggest that H(2)O(2)-induced cell damage is associated with the induction of various cell death processes that could be involved differently in plant defence reactions.     

Impact of the auxin signaling inhibitor p-chlorophenoxyisobutyric acid on short-term Cd-induced hydrogen peroxide production and growth response in barley root tip

Short-term treatment (30min) of barley roots with a low 10μM Cd concentration induced significant H(2)O(2) production in the elongation and differentiation zone of the root tip 3h after treatment. This elevated H(2)O(2) production was accompanied by root growth inhibition and probably invoked root swelling in the elongation zone of the root tip. By contrast, a high 60μM Cd concentration induced robust H(2)O(2) production in the elongation zone of the root tip already 1h after short-term treatment. This robust H(2)O(2) generation caused extensive cell death 6h after short-term treatment. Similarly to low Cd concentration, exogenously applied H(2)O(2) caused marked root growth inhibition, which at lower H(2)O(2) concentration was accompanied by root swelling. The auxin signaling inhibitor p-chlorophenoxyisobutyric acid effectively inhibited 10μM Cd-induced root growth inhibition, H(2)O(2) production and root swelling, but was ineffective in the alleviation of 60μM Cd-induced root growth inhibition and H(2)O(2) production. Our results demonstrated that Cd-induced mild oxidative stress caused root growth inhibition, likely trough the rapid reorientation of cell growth in which a crucial role was played by IAA signaling in the root tip. Strong oxidative stress induced by high Cd concentration caused extensive cell death in the elongation zone of the root tip, resulting in the cessation of root growth or even in root death.     

盐胁迫下大麦幼苗多胺的种类和状态与多胺氧化酶活性的关系

200 mmol/L的NaCl胁迫8 d大麦幼苗叶片和根系中的三种形态多胺都有不同程度地下降,其中游离态多胺含量的下降幅度最大;高氯酸不溶性结合态多胺含量变化较小.根系中PAO的活性先上升后下降,而叶片中PAO的活性先下降后上升.游离态多胺中,亚精胺和精胺(Spd Spm)的含量变化与相应部位PAO的活性变化趋势相反,表明PAO在盐胁迫下可能调节了游离态多胺的含量从而影响高氯酸可溶结合态与高氯酸不溶结合态多胺的含量.     

ABA诱导玉米叶质外体H2O2积累的机制

通过组织化学染色和电镜观察并结合酶活性分析表明,ABA可通过诱导玉米（Zea mays L、）叶片质膜NADPH氧化酶、细胞壁POD及质外体PAO活性的升高,使其质外体产生H2O2;其中质膜NADPH氧化酶起主要作用。     

Induction of polyamine oxidase 1 by Helicobacter pylori causes macrophage apoptosis by hydrogen peroxide release and mitochondrial membrane depolarization

Helicobacter pylori infects the human stomach by escaping the host immune response. One mechanism of bacterial survival and mucosal damage is induction of macrophage apoptosis, which we have reported to be dependent on polyamine synthesis by arginase and ornithine decarboxylase. During metabolic back-conversion, polyamines are oxidized and release H(2)O(2), which can cause apoptosis by mitochondrial membrane depolarization. We hypothesized that this mechanism is induced by H. pylori in macrophages. Polyamine oxidation can occur by acetylation of spermine or spermidine by spermidine/spermine N(1)-acetyltransferase prior to back-conversion by acetylpolyamine oxidase, but recently direct conversion of spermine to spermidine by the human polyamine oxidase h1, also called spermine oxidase, has been demonstrated. H. pylori induced expression and activity of the mouse homologue of this enzyme (polyamine oxidase 1 (PAO1)) by 6 h in parallel with ornithine decarboxylase, consistent with the onset of apoptosis, while spermidine/spermine N(1)-acetyltransferase activity was delayed until 18 h when late stage apoptosis had already peaked. Inhibition of PAO1 by MDL 72527 or by PAO1 small interfering RNA significantly attenuated H. pylori-induced apoptosis. Inhibition of PAO1 also significantly reduced H(2)O(2) generation, mitochondrial membrane depolarization, cytochrome c release, and caspase-3 activation. Overexpression of PAO1 by transient transfection induced macrophage apoptosis. The importance of H(2)O(2) was confirmed by inhibition of apoptosis with catalase. These studies demonstrate a new mechanism for pathogen-induced oxidative stress in macrophages in which activation of PAO1 leads to H(2)O(2) release and apoptosis by a mitochondrial-dependent cell death pathway, contributing to deficiencies in host defense in diseases such as H. pylori infection.     

Programmed cell death during embryogenesis in maize

Programmed cell death (PCD) in plants is considered an integral part of development. Evidence of DNA fragmentation, occurring at specific sites and times during embryo formation in maize (Zea mays L.), was obtained using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labelling (TUNEL) and by genomic DNA ladder detection. During the crucial period of elaboration of the primary shoot and root axis (14-20 d after pollination), TUNEL-positive nuclei are present in the scutellum, coleoptile, root cap and principally in the suspensor. Additional evidence of a form of programmed cell death occurring in these tissues comes from the detection of a DNA ladder. Upon completion of the differentiation process, all embryonic cells are TUNEL-negative, indicating that possible programmed cell death events during maize embryogenesis are confined to structures or organs that do not contribute to the adult plant body.     

Magnetic field affects meristem activity and cell differentiation in Zea mays roots

Abstract

Exposure of Zea mays seedlings to a continuous electromagnetic field (EMF) for 30 h induced a 30% stimulation in the rate of root elongation compared with the controls. It also resulted in a significant increase of cell expansion, in both the acropetal (metaxylem cell lineage) and basipetal (root cap cells) direction. In addition, in EMF-exposed roots a precocious structural disorder was observed both in differentiating metaxylem cells and root cap cells. All these features may be consistent with an advanced differentiation of root cells that are programmed to die. EMF treatment also resulted in a significant reduction in the size of the quiescent centre in the root apical meristem. The extent to which these responses are causally linked is discussed.     

Enhancement of secondary xylem cell proliferation by Arabidopsis cyclin D overexpression in tobacco plants

Secondary xylem is composed of daughter cells produced by the vascular cambium in the stem. Cell proliferation of the secondary xylem is the result of long-range cell division in the vascular cambium. Most xylem cells have a thickened secondary cell wall, representing a large amount of biomass storage. Therefore, regulation of cell division in the vascular cambium and differentiation into secondary xylem is important for biomass production. Cell division is regulated by cell cycle regulators. In this study, we confirm that cell cycle regulators influence cell division in the vascular cambium in tobacco. We produced transgenic tobacco that expresses Arabidopsis thaliana cyclin D2;1 (AtcycD2;1) and AtE2Fa-DPa under the control of the CaMV35S promoter. Each gene is a positive regulator of the cell cycle, and is known to influence the transition from G1 phase to S phase. AtcycD2;1-overexpressing tobacco had more secondary xylem cells when compared with control plants. In order to evaluate cell division activity in the vascular cambium, we prepared a Populus trichocarpa cycB1;1 (PtcycB1;1) promoter containing a destruction box motif for ubiquitination and a β-glucuronidase-encoding gene (PtcycB1;1pro:GUS). In transgenic tobacco containing PtcycB1;1pro:GUS, GUS staining was specifically observed in meristem tissues, such as the root apical meristem and vascular cambium. In addition, mitosis-monitoring plants containing AtcycD2;1 had stronger GUS staining in the cambium when compared with control plants. Our results indicated that overexpression of AtcycD enhances cell division in the vascular cambium and increases secondary xylem differentiation in tobacco. Key message We succeeded in inducing cell proliferation of cambium and enlargement of secondary xylem region by AtcycD overexpression. We also evaluated mitotic activity in cambium using cyclin-GUS fusion protein from poplar.