ROS and NO production in compatible and incompatible tomato-<Emphasis Type="Italic">Meloidogyne incognita</Emphasis> interactions

Nitric oxide (NO) has been shown to be an essential regulatory molecule in plant response to pathogen infection in synergy with reactive oxygen species (ROS). At the present, nothing is known about the role of NO in disease resistance to nematode infection. We used a resistant tomato cultivar with different sensitivity to avirulent and virulent populations of the root-knot nematode Meloidogyne incognita to investigate the key components involved in oxidative and nitrosative metabolism. We analyzed the superoxide radical production, hydrogen peroxide content, and nitric oxide synthase (NOS)-like and nitrate reductase activities, as potential sources of NO. A rapid NO accumulation and ROS production were found at 12 h after infection in compatible and incompatible tomato-nematode interactions, whereas the amount of NO and ROS gave different results 24 and 48 h after infection amongst compatible and incompatible interactions. NOS-like arginine-dependent enzyme rather than nitrate reductase was the main source of NO production, and NOS-like activity increased substantially in the incompatible interaction. We can envisage a functional overlap of both NO and ROS in tomato defence response to nematode invasion, NO and H₂O₂ cooperating in triggering hypersensitive cell death. Therefore, NO and ROS are key molecules which may help to orchestrate events following nematode challenge, and which may influence the host cellular metabolism.     

A plastidic glucose-6-phosphate dehydrogenase is responsible for hypersensitive response cell death and reactive oxygen species production

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in the supply of reduced nicotine amide cofactors for resistance to biotic and abiotic stresses. Here, we show participation of the plastidic P2 isoform of G6PDH in plant immunity. A cytosolic isoform (NbG6PDH-Cyto) and two plastidic isoforms (NbG6PDH-P1 and NbG6PDH-P2) cloned from Nicotiana benthamiana were localized in cytosol and chloroplasts, respectively. Hypersensitive response (HR) cell death and NADPH oxidase (RBOH; respiratory burst oxidase homolog)-dependent reactive oxygen species (ROS) production after recognition of INF1 elicitin, secreted by oomycete Phytophthora infestans, decreased in NbG6PDH-P2-silenced plants, but not in NbG6PDH-Cyto- and NbG6PDH-P1-silenced plants. Silencing of the cytosolic NAD kinase NbNADK1, which phosphorylates NADH to form NADPH, compromised HR cell death and ROS production, and concomitant silencing with NbG6PDH-P2 reduced HR cell death and ROS to levels near those in NbG6PDH-P2-silenced plants. Similarly, silencing NbG6PDH-P2 and NbNADK1 resulted in high susceptibility to P. infestans. These results suggest that NADPH produced by the P2 isoform of G6PDH in chloroplasts is responsible for HR cell death and ROS production mediated by RBOH and that NbNADK1 is involved in this pathway.     

小麦抗条锈反应中活性氧及细胞质钙离子的作用

 为探讨活性氧和细胞质钙离子在小麦抗条锈病反应中的作用,以小麦品种洛夫林13与具有不同致病力的单孢锈菌CY29、CY25的互作体系为平台,对锈菌侵染后小麦叶片中活性氧(ROS)积累、保护酶系(SOD、CAT和APX)活性变化动态、细胞质膜的透性改变及细胞质钙离子浓度变化做了研究。结果表明,不亲和锈菌CY25侵染可引起小麦叶片内2次ROS的爆发,第1次出现在接种后前期(接种后第2天),强度较小,第2次出现在接种后期(接种后第5天),强度较大;亲和锈菌CY29侵染只引起1次ROS爆发,出现在接种后期(接种后第5和第6天之间),但强度极大。过敏性坏死反应HR只出现在不亲和互作小麦叶片上前期ROS爆发之后,表明前期ROS爆发与HR的产生有关。伴随着后期小麦叶片中强度极高的ROS的爆发,叶片细胞原生质膜遭到了破坏,细胞内物质外渗,细胞不久便死亡,表明高强度的ROS爆发会导致细胞死亡。根据不同互作体系ROS爆发时期SOD、CAT和APX等保护酶的活性变化分析,不亲和互作体系前期强度较小的ROS爆发主要成分是H₂O₂,后期强度较高的ROS爆发主要成分是O₂^-_·和H₂O₂;亲和互作体系强度极高的ROS爆发主要成分是O₂^-_·和H₂O₂。由此说明H₂O₂是引起小麦抗病反应HR发生的因素,而O₂^-_·则是引起细胞死亡的因素。细胞质钙离子浓度变化研究表明,HR的发生与细胞质钙离子浓度增加相关。细胞质钙离子浓度的降低推迟了HR的发生,这说明小麦叶片细胞内细胞质钙离子浓度的增加是HR的必要条件,同时也说明Ca²⁺是植物HR的胞内第二信使参与植物抗病防卫反应。     

Viral infection‐induced endoplasmic reticulum stress and a membrane‐associated transcription factor NbNAC089 are involved in resistance to virus in Nicotiana benthamiana

Endoplasmic reticulum (ER) stress may induce two cell defence pathways, the unfolded protein response (UPR) or programmed cell death (PCD) upon unmitigated stress. This study confirmed that viral infection could induce ER stress through changing ER morphology and up‐regulating ER stress‐related genes, including NbNAC089. AtNAC089 serves as an ER stress sensor to regulate PCD in Arabidopsis. In this study, Nicotiana benthamiana NbNAC089 was identified. The gene encoded a 409 amino acid protein with a putative transmembrane domain near the C‐terminus and a NAC domain at the N‐terminus. NbNAC089 was localized to the ER membranes, and a truncated form of NbNAC089, lacking the transmembrane domain, was localized to the nucleus. Meanwhile, the full length of NbNAC089 was activated and cleaved in response to viral infection. The results suggest that the native protein may be translocated to the nucleus by release from the membrane during viral infection. Knock‐down of NbNAC089 in N. benthamiana increased susceptibility to Tobacco mosaic virus or Cucumber mosaic virus, and, in addition, promoted up‐regulation of UPR genes but impaired up‐regulation of PCD genes. These results show that NbNAC089 is a negative regulator of UPR and a positive regulator of PCD, and plays a role in the process of viral infection.     

拟南芥BOI 基因在抗氧化胁迫和细胞死亡中的功能分析

 本文利用BOI(Botrytis Susceptible 1 Interactor)基因敲减(knock-down) 株系、过量表达株系以及烟草瞬时表达系统,研究了BOI 基因在细胞程序化死亡(PCD)中的作用以及BOI 蛋白中RING 和WRD 结构域的功能。结果表明,BOI 基因的表达下调导致抗氧化胁迫能力下降,弱化了对活性氧介导的PCD 抑制作用;BOI 过量表达或瞬间表达可以增强对活性氧介导和α-吡啶甲酸诱导的PCD 的抑制作用。进一步研究发现,BOI 蛋白中的RING 结构域是BOI 抑制PCD 所必需的,WRD 结构域与BOI 对PCD 的抑制作用关系不大。     

Morphological and histochemical investigation of the response of Olea europaea leaves to fungal attack by Spilocaea oleagina

This study investigated the response of Olea europaea (cv. Conservolea) leaves to attack by the fungal pathogen Spilocaea oleagina. Cryostat and semithin sections of healthy and S. oleagina‐infected olive leaves were analysed histochemically for polyphenol oxidase (PPO) activity and tested for programmed cell death (PCD) induction by means of terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL). At all stages of infection, the fungus remained localized between the external and internal layers of cuticle without crossing the pectocellulosic layer. No PCD phenomena could be detected in plant cells at any stage of the disease. However, extensive degeneration of palisade parenchyma cells was observed in advanced infections, with massive loss of cytoplasmic contents and disappearance of cell compartments. Polyphenol oxidases are enzymes that, in olive, oxidize o‐diphenols (principally oleuropein and rutin) to produce o‐diquinones and melanins, substances that are toxic to many pathogens. No significant increase in overall PPO activity was found in infected leaves; on the contrary, enzyme activity was gradually lost as infection progressed, most probably due to degradation of plastids within mesophyll cells, in which such enzymes are normally confined. Only a limited local PPO activation occurred in a few upper epidermal cells of the leaf, indicating a feeble induction of a plant response.     

Growth and physiological responses of freshwater green alga Selenastrum capricornutum to allelochemical ethyl 2-methyl acetoacetate (EMA) under different initial algal densities

Most natural algicides including macrophytic allelochemicals are known to selectively inhibit algal growth. The investigations on the modes of action about the species-specific algicides are little. In this study, the effects of allelochemical ethyl 2-methyl acetoacetate (EMA) identified from reed (Phragmites communis) on the growth, physiological, and biochemical processes of green alga Selenastrum capricornutum were investigated. The results showed that EMA had multiple effects on the growth of S. capricornutum under different initial algal densities (IADs). The algal growth was inhibited by EMA at low IADs, but stimulated at high IADs. Further, the potential modes of action of EMA on S. capricornutum were explored from ultrastructure, metabolic activity, reactive oxygen species level, and lipid peroxidation to trace the microenvironment changes in the algal cells. Damage in cell structure occurred at low IAD, but cells were well developed with increased metabolic activity at high IAD. The reactive oxygen species (ROS) levels were increased under both conditions. The increase of ROS level was acute at low IAD but slow at high IAD. EMA caused significant lipid peroxidation, i.e. oxidative damage on membrane lipids at low IAD but not at high IAD. Based on these results, the initial algal density is considered an important factor to influence algal growth and physiological and biochemical responses to EMA, the effects of EMA on S. capricornutum may be “hormesis-like”, and different ROS increase ratio may be directly related with different responses of S. capricornutum to EMA.     

植物与病原物互作中的细胞程序化死亡

细胞程序化死亡(PCD)广泛存在于动植物的生长发育或抗逆过程中,而植物与病原物互作中的PCD已成为当前植物病理学的研究热点之一。本文从植物细胞程序化死亡的概念、检测方法、植物与病原物互作间的细胞程序化死亡及调控机理等方面进行了简要概述。     

活性氧参与调控植物生长发育和胁迫应激响应机理的研究进展

复杂多变的自然环境使植物进化出复杂而又巧妙的机制来感知外部信号,并对外部环境的变化做出适应性调整。在这种信号整合和决策过程中起关键作用的一类物质是活性氧(ROS)。细胞内、外产生的ROS与Ca~(2+)和电信号相互偶联组成的ROS波,向"友邻细胞"或远端细胞进行快速信号转导,进而伴随着多种信号组分共同完成应激响应表达。而ROS的产生与清除平衡关系决定了ROS在植物生长发育、器官发生和抗逆方面的作用。系统结并讨论了ROS的产生来源、信号传导机制、响应表达及ROS引起的细胞程序性死亡的最新研究进展。为深入了解ROS激活相关基因的表达,进而研究触发信号转导通路,使植物积极、高效地应对各种内在或外源的刺激信号,以调控植物的生长发育或适应胁迫环境,提供新的观点和见解。     

Differential effects of phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, and energetic metabolism inhibition on resistance of appropriate host and nonhost cereal-rust interactions

ABSTRACT Effects of phenylpropanoid and energetic metabolism inhibition on resistance were studied during appropriate host and nonhost cereal-rust interactions. In the appropriate barley-Puccinia hordei interaction, phenylalanine ammonia lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) inhibition reduced penetration resistance in two genotypes, suggesting a role for phenolics and lignins in resistance. Interestingly, penetration resistance of the barley genotype 17.5.16 was not affected by phenylpropanoid biosynthesis but penetration resistance was almost completely inhibited by D-mannose, which reduces the energy available in plant host cells. This suggests a parallel in the cellular basis of penetration resistance between 17.5.16 rust and mlo barleys powdery mildew interaction. Results revealed differing patterns of programmed cell death (PCD) in appropriate versus nonhost rust interactions. PAL and CAD inhibitors reduced PCD (hypersensitivity) in appropriate interactions. Conversely, they had no effect in PCD of wheat to P. hordei; whereas D-mannose dramatically reduced nonhost resistance and allowed colony establishment. The differential effects of inhibitors in the expression of the different resistances and the commonalities with the cereal-powdery mildew interaction is analyzed and discussed.     

Differential effects of phytotoxic metabolites from <Emphasis Type="Italic">Alternaria tagetica</Emphasis> on <Emphasis Type="Italic">Tagetes erecta</Emphasis> cell cultures

Alternaria tagetica, a fungus that causes early blight in marigold (Tagetes erecta), produces two groups of phytotoxic metabolites: one hydrophilic and the other lipophilic that show phytotoxic activity when tested by the leaf-spot assay in T. erecta. We evaluated the cellular effects of the phytotoxic culture filtrate of A. tagetica and the hydrophilic and lipophilic fractions, then determined whether the filtrate or the fractions differentially induced pathogenesis-related mechanisms in the plant. The culture filtrate and the phytotoxic fractions had adverse effects on cell viability, fresh mass, and the number of cells, and induced ROS accumulation, lipid peroxidation and DNA damage on T. erecta cell suspension cultures, and these effects are related to pathogenic mechanisms attributed to phytotoxins. However, although exposure of marigold cells to the phytotoxic culture filtrate of A. tagetica triggered programmed cell death, the hydrophilic and the lipophilic phytotoxic fractions induced death that was more related to a toxic effect leading to necrosis. This study presents a complementary perspective in the search for the roles of metabolites, including phytotoxins, produced by phytopathogenic fungi during plant infection.     

Induced cell death in Ceratocystis fimbriata by pro-apoptotic activity of a natural organic compound,perillaldehyde, through Ca2+ overload and accumulation of reactive oxygen species

Black rot caused by the fungus Ceratocystis fimbriata causes notable losses in sweet potato production. Perillaldehyde (PAE), a secondary metabolite in perilla, was studied to determine its antifungal effects on mycelial growth and spore germination of C. fimbriata. The effects of PAE on cell wall integrity and cell membrane permeability were also investigated. To elucidate the possible mechanisms of cell death triggered by PAE, sensitivity of C. fimbriata to PAE toxicity was determined by cytoplasmic and mitochondrial calcium ion concentrations ([Ca²⁺]c and [Ca²⁺]m), reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cytochrome c (cyt c) release, metacaspase activation, phosphatidylserine (PS) externalization and DNA fragmentation. The results suggest that mycelial growth and spore germination were inhibited by PAE in a dose-dependent manner. Ceratocystis fimbriata spores treated with PAE experienced dramatic Ca²⁺ overload and elevated ROS production. Compared to untreated controls, the proportion of fluorescent cells stained with the ROS indicator DCFH-DA and treated with a range of PAE concentrations from 0.0625 to 0.50 μL mL⁻¹ increased by 2.9 ± 0.79% to 27.1 ± 0.38%. Ca²⁺ overload and ROS accumulation induced depolarization of the MMP, contributing to mitochondrial dysfunction. Cyt c was released from the mitochondria to the cytosol, triggering metacaspase activation. The significant antifungal activity of PAE on C. fimbriata was demonstrated by these studies, suggesting that PAE has the potential for wide application to postharvest management of tuber crops, in addition to the application to above-ground fruit and vegetables that have been previously investigated.     

Differential necrotic lesion formation in soybean cultivars in response to soybean mosaic virus

In this study, we examined the necrosis phenotype on leaves of two cultivars of soybean (ZheA8901 and Nannong1138-2) that show varying level of resistance to soybean mosaic virus (SMV). The necrotic symptoms seen on inoculated and systemic leaves of soybean cultivar ZheA8901 were reminiscent of programmed cell death (PCD). The cell death phenotypes were evaluated using the TUNEL method, quantification of hydrogen peroxide (H₂O₂) and salicylic acid, callose production, as well as by monitoring expression of defence genes GmPR-1 and GmNPR1. Our results show that SMV inoculation induced PCD on ZheA8901 is associated with rapid increase in H₂O₂, increased SA and callose accumulation and higher defence gene expression.     

Characterization of reactive oxygen species-involved oxidative damage in Hapalosiphon species crude extract-treated wheat and onion roots

Crude extract-induced oxidative damage using the cyanobacterium, Hapalosiphon sp., was investigated in wheat seedlings ( Triticum aestivum L. cv. Norin 61) and onion seedlings ( Allium cepa L. cv. Raputa II). The analysis of root cell viability or cell death using Evans blue uptake showed that the root-tip cells of wheat and onion lost viability after 24 h and 48 h treatment with 3 g dry weight (DW) L⁻¹ of the crude extract, respectively. Lipid peroxidation was induced in the roots of both species and the shoots of onion, whereas no increase in lipid peroxide formation was observed in the wheat shoots. In onion, the degree of random DNA fragmentation increased with the increasing concentration of the extract and laddering of the DNA was observed with 6 g DW L⁻¹ of the extract, but no apparent DNA ladder formation occurred in the wheat. Pretreatment for 1 h with the NADPH oxidase inhibitors, diphenyleneidonium or imidazole, reduced the crude extract-induced root cell death in both species. From the results, we suggest that the Hapalosiphon sp. crude extract might enhance reactive oxygen species (ROS) production, which causes membrane lipid damage and fragmentation of the DNA of plant cells, resulting in cell death and growth inhibition. The crude extract-mediated phytotoxic damage might be caused by ROS, triggered by NADPH oxidase.     

<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylsphingosine,an inhibitor of sphingosine kinase,induces phytoalexin production and hypersensitive cell death of Solanaceae plants without generation of reactive oxygen species

Plant recognition of elicitors derived from pathogens induces various resistant reactions, including production of reactive oxygen species, hypersensitive cell death and accumulation of phytoalexins. Previously, we isolated a ceramide elicitor from Phytophthora infestans, which activates O₂ ⁻ production of potato suspension-cultured cells. In this study, we employed nine ceramide-related chemicals to test their elicitor activity. Although, none of the tested chemicals induced O₂ ⁻ production, N,N-dimethylsphingosine (DMS) induced accumulation of phytoalexin in potato tubers. In potato, tobacco and Nicotiana benthamiana, DMS also induced rapid cell death. DMS-treated potato cells stained with 4′,6-diamidino-2-phenylindole (DAPI) showed chromatin condensation, and isolated DNA from DMS-treated cells had ladder pattern, confirming that DMS-induced plant cell death is a hypersensitive reaction-like programmed cell death. Involvement of ceramide signaling in induction of plant defense reactions is discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Programmed cell death pathways induced by early plant‐virus infection are determined by isolate virulence and stage of infection

Programmed cell death (PCD) pathways caused by Turnip mosaic virus (TuMV) infection before symptom appearance were studied by light microscopy and electrolyte leakage following sap inoculation of Brassica carinata (Ethiopian mustard) TZ‐SMN‐44‐6 plants. Leaf responses to inoculation with avirulent (TuMV‐avir) and virulent (TuMV‐vir) isolates, and mock‐inoculation, were compared at 2, 20 and 52 h after inoculation (hai). The phenotypes induced were localized resistance (TuMV‐avir) and systemic susceptibility (TuMV‐vir). No visible TuMV symptoms were recorded in any inoculated plants during the 2–52 hai sampling period, but appeared as chlorotic spots in inoculated leaves at 5 days after inoculation. With TuMV‐vir alone, they were followed by systemic infection (mosaic). Dead cell number, deformation, percentage area and percentage integrated intensity, and conductivity of electrolyte leakage data, were analysed to examine their possible roles in stimulating cell death pathways. At 2 hai, dead cell number and percentage area were significantly greater for TuMV‐avir than TuMV‐vir infection or mock‐inoculation. Overall, isolate TuMV‐vir caused significantly greater cell deformation than TuMV‐avir, whereas wounding by mock‐inoculation had negligible effects. By 52 hai, isolate TuMV‐avir caused significantly greater electrolyte leakage than isolate TuMV‐vir or mock‐inoculation. This suggests both isolates triggered morphological changes consistent with apoptotic‐like PCD and necrosis‐like PCD that depended upon isolate virulence and stage of infection, respectively. These findings highlight how quantification of dead cell deformation and electrolyte leakage offer a new understanding of compatible and incompatible plant responses to early virus infection in plants.     

<Emphasis Type="Italic">Fusarium</Emphasis> elicitor-dependent calcium influx and associated ros generation in tomato is independent of cell death

Fusarium oxysporum f. sp. lycopersici elicitor (EFOL-2) treatment induces cytosolic influx of calcium in Fusarium-resistant tomato suspension culture. The calcium signature was found to be biphasic, which is characteristic of recognition of oligosaccharides in the elicitor preparation. Further, several lines of evidence such as, (i) attainment of saturation level of the [Ca²⁺]_cyt at a definite extra-cellular calcium concentration (ii) prominent reduction in EFOL-2-induced influx in [Ca²⁺]_cyt on treatment with the calcium channel blockers verapamil and diltiazem and (iii) establishment of a refractory stage of [Ca²⁺]_cyt level upon repeated stimulation by EFOL-2, is indicative of receptor-mediated activation of the calcium channel for cytosolic elevation. In addition, inhibition of EFOL-2-induced [Ca²⁺]_cyt increase by protein kinase inhibitor staurosporine and wortmannin indicate phosphorylation is a regulatory event of calcium influx. Additionally, monitoring of cell death on EFOL-2 treatment indicated that the degree of ROS generation is not capable of inducing cell death. Inhibition of ROS generation on two separate occasions such as, calcium-free media and on treatment with inhibitors causing calcium channel occlusion revealed ROS generation as a successive event of calcium influx.     

Early Events During Quiescent Infection Development by Colletotrichum gloeosporioides in Unripe Avocado Fruits

Inoculation of avocado pericarp tissue with Colletotrichum gloeospori-oides and treatment of avocado cell cultures with the cell wall elicitor of C. gloeosporioidesboth increased the production of reactive oxygen species (ROS). However, whereas the production of ROS could be detected within minutes in avocado cell suspensions, it was detected only after 2 h following inoculation of pericarp tissue. Protein kinase inhibitors such as K-252a and staurosporine and the phosphatase inhibitor microcystin-LR inhibited the release of H(2)O(2) from avocado cell suspensions. When 1 mM H(2)O(2) was exogenously applied to pericarp tissue, it enhanced ROS, phenyl-alanine ammonia lyase (PAL) activity, and epicatechin levels. But, when H(2)O(2) treatment was applied following staurosporine treatment, PAL activity was no longer induced. The uninduced ROS production in pericarp tissue of freshly harvested, unripe, resistant fruit was twice as high as in ripe, susceptible fruit. Challenge inoculation of resistant fruit further increased the ROS level; however, this increase did not occur in susceptible fruits. The current findings are consistent with the hypothesis that production of ROS is induced by fungal infection of unripe fruits and, consequently, may modulate resistance, resulting in the inhibition of fungal development and quiescence.