Harpin inactivates mitochondria in Arabidopsis suspension cells

Harpin is a well-known proteinaceous bacterial elicitor that can induce an oxidative burst and programmed cell death in various host plants. Given the demonstrated roles of mitochondria in animal apoptosis, we investigated the effect of harpin from Pseudomonas syringae on mitochondrial functions in Arabidopsis suspension cells in detail. Fluorescence microscopy in conjunction with double-staining for reactive oxygen species (ROS) and mitochondria suggested co-localization of mitochondria and ROS generation. Plant defense responses or cell death after pathogen attack have been suggested to be regulated by the concerted action of ROS and nitric oxide (NO). However, although Arabidopsis cells respond to harpin treatment with NO generation, time course analyses suggest that NO generation is not involved in initial responses but, rather, is a consequence of cellular decay. Among the fast responses we observed was a decrease of the mitochondrial membrane potential deltapsim, and, possibly as a direct consequence, of ATP production. Furthermore, treatment of Arabidopsis cells with harpin protein induced a rapid cytochrome C release from mitochondria into the cytosol, which is regarded as a hallmark of programmed cell death or apoptosis. Northern and DNA array analyses showed strong induction of protecting or scavenging systems such as alternative oxidase and small heat shock proteins, components that are known to be associated with cellular stress responses. In sum, the presented data suggest that harpin inactivates mitochondria in Arabidopsis cells.     

Hydrogen Peroxide from the Oxidative Burst Is Neither Necessary Nor Sufficient for Hypersensitive Cell Death Induction, Phenylalanine Ammonia Lyase Stimulation, Salicylic Acid Accumulation, or Scopoletin Consumption in Cultured Tobacco Cells Treated with Elicitin

H(2)O(2) from the oxidative burst, cell death, and defense responses such as the production of phenylalanine ammonia lyase (PAL), salicylic acid (SA), and scopoletin were analyzed in cultured tobacco (Nicotiana tabacum) cells treated with three proteinaceous elicitors: two elicitins (alpha-megaspermin and beta-megaspermin) and one glycoprotein. These three proteins have been isolated from Phytophthora megasperma H20 and have been previously shown to be equally efficient in inducing a hypersensitive response (HR) upon infiltration into tobacco leaves. However, in cultured tobacco cells these elicitors exhibited strikingly different biological activities. beta-Megaspermin was the only elicitor that caused cell death and induced a strong, biphasic H(2)O(2) burst. Both elicitins stimulated PAL activity similarly and strongly, while the glycoprotein caused only a slight increase. Only elicitins induced SA accumulation and scopoletin consumption, and beta-megaspermin was more efficient. To assess the role of H(2)O(2) in HR cell death and defense response expression in elicitin-treated cells, a gain and loss of function strategy was used. Our results indicated that H(2)O(2) was neither necessary nor sufficient for HR cell death, PAL activation, or SA accumulation, and that extracellular H(2)O(2) was not a direct cause of intracellular scopoletin consumption.     

Light and oxygen are not required for harpin-induced cell death

Nicotiana sylvestris leaves challenged by the bacterial elicitor harpin N(Ea) were used as a model system in which to determine the respective roles of light, oxygen, photosynthesis, and respiration in the programmed cell death response in plants. The appearance of cell death markers, such as membrane damage, nuclear fragmentation, and induction of the stress-responsive element Tnt1, was observed in all conditions. However, the cell death process was delayed in the dark compared with the light, despite a similar accumulation of superoxide and hydrogen peroxide in the chloroplasts. In contrast, harpin-induced cell death was accelerated under very low oxygen (<0.1% O(2)) compared with air. Oxygen deprivation impaired accumulation of chloroplastic reactive oxygen species (ROS) and the induction of cytosolic antioxidant genes in both the light and the dark. It also attenuates the collapse of photosynthetic capacity and the respiratory burst driven by mitochondrial alternative oxidase activity observed in air. Since alternative oxidase is known to limit overreduction of the respiratory chain, these results strongly suggest that mitochondrial ROS accumulate in leaves elicited under low oxygen. We conclude that the harpin-induced cell death does not require ROS accumulation in the apoplast or in the chloroplasts but that mitochondrial ROS could be important in the orchestration of the cell suicide program.     

Elicitation of the Hypersensitive Responses in Tabacco by a10.6 kD Proteinaceous Elicitor from Phytophthora palmi

A 10.6 kD heat resistant, proteinacious elicitor was purified from the culture filtrate of Phytophthora palmivora Butler but not from P. melonis Katsura. The 10.6 kD elicitor is a holoprotein devoid of glycoside. It can cause hypersensitive necrosis of the detached tobacco ( Nicotiatna tabacum L. ) leaves 48 h post-inoculation with dosages of above 40 μg. Four cell types were investigated by using Confocal microscopy, and 2' ,7'-dichlorodihydrofluorescein diacetate (DCFDA) as a probe of H2O2 production. It was showed that oxidative burst occurred in cultured suspension cells as well as in mesophyll cells, epidermal cells and guard cells within 10 min upon the elicitor treatment. The hypersensitive cell death appeared 6 h after the treatment when inoculated with fluorescein diacetate (FDA) as indicator of cell viability. These results suggest that H2O2 accumulation was the main cause of the hypersensitive cell death in tobacco induced by the 10.6 kD elicitor. This 10.6 kD elicitor may belong to the family of elicitins.     

Phytophthora palmi分泌的10.6kD蛋白激发烟草的过敏反应

从疫霉菌Ｐｈｙｔｏｐｈｔｈｏｒａ ｐａｌｍｉｖｏｒａ Ｂｕｔｌｅｒ的培养滤液中分离出分子量为１０．６ｋＤ的不含糖基的耐热蛋白．这种１０．６ｋ蛋白能诱导烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）叶片发生过敏性坏死反应。而疫霉菌另一种Ｐ．ｍｅｌｏｎｉｓ Ｋａｔｓｕｒａ的培养滤液中不含这种类似蛋白,不能诱导烟草叶片发生过敏反应。利用共聚焦激光扫描显微镜,以荧光探剂ＦＤＡ（ｆｌｕｏｒｅｓｃｅｉｎ ｄ     

Defence gene expression in soybean is linked to the status of the cell death program

Soybean cell cultures (cv. Williams 82) respond to Pseudomonas syringae bacteria expressing the avirulence gene AvrA with a hypersensitive reaction, a programmed cell death (PCD) of plant cells to pathogen attack. This PCD is under control of salicylic acid (SA) via an unknown mechanism. In the presence of low concentrations of SA, the cells undergo a very rapid cell death, which needs only half of the time required for the normal hypersensitive reaction (HR). Northern blot studies for defence-related genes show that the expression of many of these genes is tightly linked to the status of the cell death program rather than to pathogen-derived elicitors. Thus the expression is much faster in the SA-accelerated PCD than in the normal hypersensitive reaction. In contrast, other pathogen-responsive genes are induced independently of the speed of PCD, indicating a divergent signalling mechanism. The production of reactive oxygen species during the oxidative burst of bacteria-inoculated soybean cells is slightly enhanced in the presence of SA but occurs at the same time as in untreated cells, suggesting that SA exhibits the control of the PCD downstream of the oxidative burst. Consistent with these findings a HR-specific marker gene is neither directly induced by H₂O₂ or SA. However, this gene shows a high expression in the regular HR and is induced much faster in the SA-accelerated PCD.     

Harpin,An Elicitor of the Hypersensitive Response in Tobacco Caused by Erwinia amylovora,Elicits Active Oxygen Production in Suspension Cells

Active oxygen (AO) production and a K+/H+ exchange response (XR) are two concurrent early events associated with incompatible plant-bacteria interactions that result in a hypersensitive response (HR). Recently, a protein, termed harpin, produced by Erwinia amylovora has been reported to be the elicitor responsible for the HR caused by this pathogen. Although both the bacterium and harpin are reported to induce XR in tobacco (Nicotiana tabacum) cell suspensions, there have been no reports regarding the concurrent production of AO in this system. Here we report that E. amylovora stimulates the AO response, whereas an E. amylovora mutant that does not produce harpin does not elicit the AO response. In addition, a cell-free preparation of harpin induces AO production. This study indicates that harpin may be the bacterial elicitor of the XR and AO responses during the development of E. amylovora-induced HR.     

Palmin诱导烟草氧化猝发的机制

为探讨植物对病原微生物的防御机制和激发子启动植物体内的信号转导应答过程,本文研究了Phytophthora palmi激发子palmin诱导其非寄主亲和性烟草的叶片和悬浮细胞系产生氧化猝发的分子机理.利用生化分析和激光共聚焦显微扫描技术动态观察palmin诱导烟草过敏反应中O*-2和H2O2的形成、胞间转移及引起细胞死亡的特性.结果表明:palmin诱导激活了烟草细胞内NADPH氧化酶,产生大量的O*-2;O*-2在SOD催化下迅速转变成H2O2,并且H2O2在一定范围的细胞间转移和积累,最后诱发烟草细胞的过敏性坏死反应.palmin诱导氧化猝发过程还有Ca2+和蛋白激酶的参与.     

Thaxtomin A induces programmed cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> suspension-cultured cells

Thaxtomin A is the main phytotoxin produced by Streptomyces scabiei, the causative agent of common scab disease of potato. Pathogenicity of S. scabiei is dependent on the production of thaxtomin A which is required for the development of disease symptoms, such as growth inhibition and cell death. We investigated whether thaxtomin A-induced cell death was similar to the hypersensitive cell death that often occurs in response to specific pathogens or phytotoxins during the so-called hypersensitive response (HR). We demonstrated that thaxtomin A induced in Arabidopsis thaliana suspension-cultured cells a genetically controlled cell death that required active gene expression and de novo protein synthesis, and which involved fragmentation of nuclear DNA, a characteristic hallmark of apoptosis. The thaxtomin A-induced form of programmed cell death (PCD) was not a typical HR, since defence responses generally preceding or associated with the HR, such as rapid medium alkalization, oxidative burst and expression of defence-related genes PR1 and PDF1.2, were not observed in plant cells following addition of thaxtomin A. Thaxtomin A has been shown to inhibit cellulose biosynthesis (Scheible et al. in Plant Cell 15:1781, 2003). We showed that isoxaben, a specific inhibitor of cellulose biosynthesis, also induced in Arabidopsis cell suspensions a PCD similar to that induced by thaxtomin A. These data suggested that rapid changes in the plant cell wall composition and organization can induce PCD in plant cells. We discuss how rapid inhibition of cellulose biosynthesis may trigger this process.     

激发子α-吡啶羧酸诱发拟南芥和水稻悬浮细胞中依赖于水杨酸、茉莉酸/乙烯和钙离子途径的防卫反应

α-吡啶羧酸(PA)是动物细胞程序化死亡的诱导物.我们前期的研究表明,PA可以激发单子叶模式植物水稻的过敏反应(HR).进一步用双子叶模式植物拟南芥(Arabidopsis thaliana)进行的研究表明,PA是一个广谱的植物HR反应的激发子,包括诱导氧进发和细胞死亡.我们探究了PA诱导的拟南芥防卫反应途径,利用不同信号途径标志基因PR-1,PR-2和PDF1.2受诱导剂量和时间激活的结果,表明PA可以同时激活水杨酸和茉莉酸/乙烯依赖的防卫途径.我们也发现PA诱导水稻悬浮细胞产生活性氧是钙离子依赖性的.综合所有结果,我们认为PA可以作为一个非专化性的植物防卫反应激发子,可望用于系统获得性抗性激发的细胞模型的建立.     

激发子α-吡啶羧酸诱发拟南芥和水稻悬浮细胞中依赖于水杨酸、茉莉酸/乙烯和钙离子途径的防卫反应(英文)

α-吡啶羧酸(PA)是动物细胞程序化死亡的诱导物。我们前期的研究表明,PA可以激发单子叶模式植物水稻的过敏反应(HR)。进一步用双子叶模式植物拟南芥(Arabidopsis thaliana)进行的研究表明,PA是一个广谱的植物HR反应的激发子,包括诱导氧进发和细胞死亡。我们探究了PA诱导的拟南芥防卫反应途径,利用不同信号途径标志基因PR-1,PR-2和PDF1.2受诱导剂量和时间激活的结果,表明PA可以同时激活水杨酸和茉莉酸/乙烯依赖的防卫途径。我们也发现PA诱导水稻悬浮细胞产生活性氧是钙离子依赖性的。综合所有结果,我们认为PA可以作为一个非专化性的植物防卫反应激发子,可望用于系统获得性抗性激发的细胞模型的建立。     

Harpin modulates the accumulation of salicylic acid by Arabidopsis cells via apoplastic alkalization

It is reported here that salicylic acid (SA) is rapidly taken up by Arabidopsis cells, and its uptake is accompanied by media alkalization and cytosolic acidification, and it is inhibited by the ionophore nigericin, suggesting that its import is linked with that of H+ and driven by a proton gradient. Such import and accumulation declined sharply within a narrow physiological pH range (pH 5.7-6.1), corresponding to a reduction in the [H+] of the media from 1.99 micromol l(-1) to 0.79 micromol l(-1). Following the initial uptake, SA was exported back into the media as free SA against a continued [H+]-dependent import. Since the uptake and accumulation of SA declines sharply within a narrow pH range and cell wall alkalization is an early response during incompatible plant/pathogen interactions, the bacterial elicitor harpin(Pss) was used to investigate how SA transport may be modulated during defence responses. Harpin induced a rapid and sustained alkalization of the cell suspension media, reaching the critical pH (pH 5.9-6.1) at which SA import is inhibited at c. 60 min. Such media alkalization corresponded with a reduction in the SA associated with cells co-treated with harpin, and an inhibition of SA uptake in cells pretreated with harpin. Scavengers of ROS, or compounds which generate H2O2 or NO had little effect on the import or net export of SA, suggesting that media alkalization induced by harpin is sufficient to modulate the kinetics of SA transport.     

Transgenic plant cells lacking mitochondrial alternative oxidase have increased susceptibility to mitochondria-dependent and -independent pathways of programmed cell death

The plant mitochondrial electron transport chain is branched such that electrons at ubiquinol can be diverted to oxygen via the alternative oxidase (AOX). This pathway does not contribute to ATP synthesis but can dampen the mitochondrial generation of reactive oxygen species. Here, we establish that transgenic tobacco (Nicotiana tabacum L. cv Petit Havana SR1) cells lacking AOX (AS8 cells) show increased susceptibility to three different death-inducing compounds (H(2)O(2), salicylic acid [SA], and the protein phosphatase inhibitor cantharidin) in comparison with wild-type cells. The timing and extent of AS8 cell death are very similar among the three treatments and, in each case, are accompanied by the accumulation of oligonucleosomal fragments of DNA, indicative of programmed cell death. Death induced by H(2)O(2) or SA occurs by a mitochondria-dependent pathway characterized by cytochrome c release from the mitochondrion. Conversely, death induced by cantharidin occurs by a pathway without any obvious mitochondrial involvement. The ability of AOX to attenuate these death pathways may relate to its ability to maintain mitochondrial function after insult with a death-inducing compound or may relate to its ability to prevent chronic oxidative stress within the mitochondrion. In support of the latter, long-term treatment of AS8 cells with an antioxidant compound increased the resistance of AS8 cells to SA- or cantharidin-induced death. The results indicate that plants maintain both mitochondria-dependent and -independent pathways of programmed cell death and that AOX may act as an important mitochondrial "survival protein" against such death.     

A loss of resistance to avirulent bacterial pathogens in tobacco is associated with the attenuation of a salicylic acid-potentiated oxidative burst

The role of salicylic acid (SA) in events occurring before cell death during the hypersensitive reaction (HR) was investigated in leaves of wild-type tobacco Samsun NN and in transgenic lines expressing salicylate hydroxylase (35S-SH-L). Challenge of 35S-SH-L tobacco with avirulent strains of Pseudomonas syringae gave rise to symptoms resembling those normally associated with a compatible response to virulent strains in terms of visible phenotype, kinetics of bacterial multiplication, and escape from the infection site. Compared with responses in wild-type tobacco, both the onset of plant cell death and the induction of an active oxygen species-responsive promoter (AoPR1-GUS) were delayed following challenge of 35S-SH-L plants with avirulent bacteria. The oxidative burst occurring after challenge with avirulent bacteria was visualized histochemically and quantified in situ. H2O2 accumulation at reaction sites was evident within 1 h after inoculation in wild-type tobacco, whereas in 35S-SH-L plants the onset of H2O2 accumulation was delayed by 2-3 h. The delay in H2O2 generation was correlated with a reduction in the transient rise in SA that usually occurred within 1-2 h following inoculation in wild-type plants. Our data indicate that an early transient rise in SA potentiates the oxidative burst, with resultant effects on accumulation of H2O2, plant cell death and also defence-gene induction, factors that together may determine the outcome of plant-pathogen interactions.     

Evidence of mitochondrial involvement in the transduction of signals required for the induction of genes associated with pathogen attack and senescence

Using the mRNA differential display technique, seven cDNAs have been isolated that are rapidly induced when cultured tobacco (Nicotiana tabacum) cells are treated with the mitochondrial electron transport inhibitor antimycin A (AA). Interestingly, six of the cDNAs show distinct similarity to genes known to be induced by processes that involve programmed cell death (PCD), such as senescence and pathogen attack. All of the cDNAs as well as Aox1, a gene encoding the alternative oxidase, were found to also be strongly induced by H2O2 and salicylic acid (SA). AA, H2O2 and SA treatment of tobacco cells caused a rapid rise in intracellular ROS accumulation that, when prevented by antioxidant treatment, resulted in inhibition of gene induction. Besides AA, both H2O2 and SA were found to disrupt normal mitochondrial function resulting in decreased rates of electron transport and a lowering of cellular ATP levels. Furthermore, the pre-treatment of tobacco cells with bongkrekic acid, a known inhibitor of the mitochondrial permeability transition pore in animal cells, was found to completely block gene induction when AA, H2O2 or SA were subsequently added. These findings suggest that the mitochondrion may serve an important role in conveying intracellular stress signals to the nucleus, leading to alterations in gene expression.     

The elicitor cryptogein blocks glucose transport in tobacco cells

Cryptogein is a 10-kD protein secreted by the oomycete Phytophthora cryptogea that induces a hypersensitive response on tobacco (Nicotiana tabacum var. Xanthi) plants and a systemic acquired resistance against various pathogens. The mode of action of this elicitor has been studied using tobacco cell suspensions. Our previous data indicated that within minutes, cryptogein signaling involves various events including changes in ion fluxes, protein phosphorylation, sugar metabolism, and, eventually, cell death. These results suggested that transport of sugars could be affected and, thus, involved in the complex relationships between plant and microorganisms via elicitors. This led us to investigate the effects of cryptogein on glucose (Glc) uptake and mitochondrial activity in tobacco cells. Cryptogein induces an immediate inhibition of Glc uptake, which is not attributable to plasma membrane (PM) depolarization. Conversely, cryptogein-induced valine uptake is because of PM depolarization. Inhibition of the PM Glc transporter(s) was shown to be mediated by a calcium-dependent phosphorylation process, and is independent of active oxygen species production. This inhibition was associated with a strong decrease in O(2) uptake rate by cells and a large mitochondrial membrane depolarization. Thus, inhibition of Glc uptake accompanied by inhibition of phosphorylative oxidation may participate in hypersensitive cell death. These results are discussed in the context of competition between plants and microorganisms for apoplastic sugars.     

Lack of respiratory chain complex I impairs alternative oxidase engagement and modulates redox signaling during elicitor-induced cell death in tobacco

Alternative oxidase (AOX) functions in stress resistance by preventing accumulation of reactive oxygen species (ROS), but little is known about in vivo partitioning of electron flow between AOX and the cytochrome pathway. We investigated the relationships between AOX expression and in vivo activity in Nicotiana sylvestris and the complex I-deficient CMSII mutant in response to a cell death elicitor. While a specific AOX1 isoform in the active reduced state was constitutively overexpressed in CMSII, partitioning through the alternative pathway was similar to the wild type. Lack of correlation between AOX content and activity indicates severe metabolic constraints in nonstressed mutant leaves. The bacterial elicitor harpin N(Ea) induced similar timing and extent of cell death and a twofold respiratory burst in both genotypes with little change in AOX amounts. However, partitioning to AOX was increased twofold in the wild type but remained unchanged in CMSII. Oxidative phosphorylation modeling indicated a twofold ATP increase in both genotypes. By contrast, mitochondrial superoxide dismutase activity and reduced forms of ascorbate and glutathione were higher in CMSII than in the wild type. These results demonstrate genetically programmed flexibility of plant respiratory routes and antioxidants in response to elicitors and suggest that sustained ATP production, rather than AOX activity by itself or mitochondrial ROS, might be important for in planta cell death.