Overexpression of FBR41 enhances resistance to sphinganine analog mycotoxin‐induced cell death and Alternaria stem canker in tomato

Fumonisin B1 (FB1) and Alternaria alternate f. sp. lycopersici (AAL)‐toxin are classified as sphinganine analog mycotoxins (SAMTs), which induce programmed cell death (PCD) in plants and pose health threat to humans who consume the contaminated crop products. Herein, Fumonisin B1 Resistant41 (FBR41), a dominant mutant allele, was identified by map‐based cloning of Arabidopsis FB1‐resistant mutant fbr41, then ectopically expressed in AAL‐toxin sensitive tomato (Solanum lycopersicum) cultivar. FBR41‐overexpressing tomato plants exhibited less severe cell death phenotype upon AAL‐toxin treatment. Analysis of free sphingoid bases showed that both fbr41 and FBR41‐overexpressing tomato plants accumulated less sphinganine and phytosphingosine upon FB1 and AAL‐toxin treatment, respectively. Alternaria stem canker is a disease caused by AAL and responsible for severe economic losses in tomato production, and FBR41‐overexpressing tomato plants exhibited enhanced resistance to AAL with decreased fungal biomass and less cell death, which was accompanied by attenuated accumulation of free sphingoid bases and jasmonate (JA). Taken together, our results indicate that FBR41 is potential in inhibiting SAMT‐induced PCD and controlling Alternaria stem canker in tomato.     

细胞色素c能诱导植物细胞编程性死亡

以悬浮培养的胡萝卜（ＤａｕｃｕｓｃａｒｏｔａＬ．）与烟草（ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ．ｃｖ．ＢＹ２）细胞原生质体为材料,加入一定浓度的细胞色素ｃ和ｄＡＴＰ。不同取样时间的ＤＡＰＩ荧光染色与电镜超薄切片观察的结果显示染色质发生凝集、趋边化,最终形成凋亡小体。核酸电泳显示ＤＮＡ发生特异降解并形成电泳“阶梯”（ＤＮＡｌａｄｄｅｒ）。用末端脱氧核糖核酸转移酶介导的ｄＵＴＰ切口末端标记方法（ＴＵＮＥＬ）检测发现ＤＮＡ的３＇ＯＨ断端被原位特异标记。以上结果说明：细胞色素ｃ能诱导植物细胞发生典型的凋亡。     

Most AAL toxin-sensitive Nicotiana species are resistant to the tomato fungal pathogen Alternaria alternata f. sp. lycopersici

The phytopathogenic fungus Alternaria alternata f. sp. lycopersici produces AAL toxins required to colonize susceptible tomato (Lycopersicon esculentum) plants. AAL toxins and fumonisins of the unrelated fungus Fusarium moniliforme are sphinganine-analog mycotoxins (SAMs), which are toxic for some plant species and mammalian cell lines. Insensitivity of tomato to SAMs is determined by the Alternaria stem canker gene 1 (Asc-1), and sensitivity is associated with a mutated Asc-1. We show that SAM-sensitive species occur at a low frequency in the Nicotiana genus and that candidate Asc-1 homologs are still present in those species. In Nicotiana spp., SAM-sensitivity and insensitivity also is mediated by a single codominant locus, suggesting that SAM-sensitive genotypes are host for A. alternata f. sp. lycopersici. Nicotiana umbratica plants homozygous for SAM-sensitivity are indeed susceptible to A. alternata f. sp. lycopersici. In contrast, SAM-sensitive genotypes of Nicotiana spegazzinii, Nicotiana acuminata var. acuminata, Nicotiana bonariensis, and Nicotiana langsdorffii are resistant to A. alternata f. sp. lycopersici infection concomitant with localized cell death. Additional (nonhost) resistance mechanisms to A. alternata f. sp. lycopersici that are not based on an insensitivity to SAMs are proposed to be present in Nicotiana species.     

Mycotoxins reveal connections between plants and animals in apoptosis and ceramide signaling

Plants undergo programmed cell death during development and disease in contexts that are functionally analogous to apoptosis in animals. Recent studies involving plant cell death induced by mycotoxins, pathogens and lethal mutations along with the cell-autonomous death during development now point to several conserved connections to apoptosis in animals. Morphological markers indicative of apoptosis recently reported in plants include TUNEL positive cells, DNA ladders, Ca2+-activated nucleosomal DNA cleavage, and formation of apoptotic-like bodies that occur in some but not all situations involving ordered cell death. In parallel studies with animal and plant cells treated with sphinganine analog mycotoxins our results indicate that the induction and inhibition of death may be mediated by ceramide-linked signaling systems. The presence and significance of ceramide-linked second messenger systems is well documented in animals but is virtually unknown in plants. Further research will discern the manner in which the important function of programmed cell death is conserved as well as diverged between the two kingdoms.     

Direct detection and quantification of Alternaria alternata lycopersici toxins using high-performance liquid chromatography-evaporative light-scattering detection

Alternaria alternata lycopersici toxins are produced by the tomato pathogenic fungus A. alternata f. sp. lycopersici. They are primary determinants for stem canker disease of tomato and induce apoptosis in animals and plants. Due to the lack of a UV chromophore, their detection has relied on derivatizations or immunoassays, which require tedious pre-assay processes. Here, we describe a method for direct and quantitative detection of the toxins. The method employs an evaporative light-scattering detector (ELSD) coupled to HPLC. It gives a linear response in the range of 400-4000 ng toxin, with a detection limit of about 6 ng/mul. The detection of all major toxin congeners by HPLC-ESLD was confirmed by liquid chromatography-mass spectrometry. Thus, the method provides a simple and sensitive tool for the studies of this group of host-specific mycotoxins.     

Overexpression of the tomato Asc-1 gene mediates high insensitivity to AAL toxins and fumonisin B1 in tomato hairy roots and confers resistance to Alternaria alternata f. sp. lycopersici in Nicotiana umbratica plants

The sphinganine-analog mycotoxins (SAMs) fumonisin B1 and AAL toxins are inhibitors of eukaryotic sphinganine N-acyltransferase in vitro. Treatment of eukaryotes with SAMs generally results in an accumulation of sphingoid base precursors and a depletion of complex sphingolipids. The asc,asc genotypes of tomato (Lycopersicon esculentum) and Nicotiana umbratica are sensitive to SAMs and host of the AAL toxin-producing fungus Alternaria alternata f. sp. lycopersici. Codominant insensitivity to SAMs in tomato is mediated by the Asc-1 gene, and sensitivity is associated with a frame-shift mutation present in asc-1. We investigated the function of Asc-1 in mediating insensitivity to SAMs and resistance to the fungus by overexpression of asc-1 and Asc-1. In this study, it is shown that overexpression of these genes did not lead to visual symptoms in tomato hairy roots and N. umbratica plants. Overexpression of asc-1 did not influence the (in)sensitivity to SAMs. Overexpression of Asc-1 in SAM-sensitive hairy roots and N. umbratica plants, however, mediated a high insensitivity to SAMs and resistance to plant infection by Alternaria alternata f. sp. lycopersici.     

Effect of ethrel on apoptosis in carrot protoplasts

In recent years, apoptosis has been reported to exist in plants during normal development and in response to stress. However, little is known about the relation of hormones to this form of programmed cell death. Here, we report examination of characteristics of apoptosis in carrot protoplasts induced by ethylene evolved from ethrel (2-chloroethylphosphonic acid). Nucleus condensation and DNA ladders were observed, and neutral comet assay, which detects DNA cleavage, also provided evidence that ethrel treatment resulted in nuclear DNA fragmentation. Strikingly, a close correlation between the incidence of DNA comets and the percentage of apoptotic protoplasts was shown in ethrel-treated carrot protoplasts. In conclusion, this study demonstrated that ethylene is an active inducer of apoptosis in carrot protoplasts, and that ethylene-induced plant cell death showed characteristics similar to those of apoptosis in animals.     

Genetic and physical analysis of a YAC contig spanning the fungal disease resistance locus Asc of tomato (Lycopersicon esculentum)

The Alternaria stem canker disease of tomato is caused by the necrotrophic fungal pathogen Alternaria alternata f. sp. lycopersici (AAL). The fungus produces AAL toxins that kill the plant tissue. Resistance to the fungus segregates as a single locus, called Asc, and has been genetically mapped on chromosome 3 of tomato. We describe here the establishment of a 1383-kb YAC contig covering the Asc locus and a series of plants selected for recombination events around the Asc locus. It was shown that the YAC contig corresponds to a genetic distance of at least 11.2 cM. Thus, the recombination rate in the Asc region is six times higher (123 kb/cM) than the average for the tomato genome. Furthermore, the Asc locus could be localised to a 91-kb fragment, thus paving the way for the cloning and identification of the Asc gene(s) by complementation. Received: 31 July 1998 / Accepted: 6 October 1998     

Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation

Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevent PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmark of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. CA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.     

AAL Toxins,funionisms (biology and chemistry) and host-specificity concepts

The AAL toxins and the fumonisins (FB₁ and FB₂) are structurally related and produced respectively by Alternaria alternata f.sp. lycopersici and Fusarium moniliforme. AAL toxin is characterized as a hostspecific toxin, toxic to tomato, whereas fumonisin B₁ causes equine leukoencephalomalacia. FB₁ and FB₂ were biologically active in susceptible tomato tissue (Earlypak-7) and animal tissue culture (rat hepatoma H4TG and dog kidney MDCK). Conversely, AAL toxin was also active in the rat and dog tissue culture cells. Both fungi produce toxin/s in culture that cause death in rats; these toxins are other than AAL and fumonisin. The peracetylated derivatives of AAL and FB₁ are biologically inactive in both the tomato bioassay and the animal tissue culture systems. Acetylation of the amine renders AAL inactive. The hydrolysis product of AAL (pentolamine) is toxic to the susceptible tomato line whereas the pentolamine of fumonisin is not.AAL and FB₁ can be analyzed by Continuous Flow Fast Atom Bombardment (CFFAB) and Ionspray Mass Spectrometry (ISM), both sensitive to the picomole range. The N-acetyl of the TFA hydrolysis product of AAL and FB₁ is determined by comparing the fragment ions at m/z 86 and 140 for FB₁ and 72 and 126 for AAL.Published as paper No. 19,598 of the contribution series of the Minnesota Agricultural Experiment Station based on research conducted under Project 22–34H, supported by HATCH funds.     

羟自由基诱导的烟草原生质体的凋亡:流式细胞法的新证据

用1.0 mmol/L FeSO4/0.5 mmol/L H202处理烟草(Nicotiana tabacum L.cultivar BY 2)原生质体,发现羟自由基能够诱导烟草原生质体的凋亡.具体表现为细胞核皱缩、DNA Ladder、TUNEL阳性反应等典型的凋亡特征.在动物细胞凋亡过程中,线粒体起着非常重要的作用,其中膜电位(△ψm)的变化以及由其引起的位于线粒体膜上的通透性孔(PTP)的开放与Cyt c的释放有关.另外,在动物凋亡细胞中,磷脂酰丝氨酸(phosphatidyl serine,PS)会从细胞膜内侧向外翻转.为了判断植物细胞凋亡过程中膜电位的变化情况以及PS的外翻程度,我们采用了流式细胞法.结果表明,随着处理时间的延长,烟草原生质体线粒体的膜电位逐渐降低;膜内PS大量外翻.说明由羟自由基和烟草原生质体组成的凋亡体系是一种可靠的凋亡组合,可以用来对植物细胞凋亡机理做进一步研究.     

Programmed cell death in cereal aleurone

Progress in understanding programmed cell death (PCD) in the cereal aleurone is described. Cereal aleurone cells are specialized endosperm cells that function to synthesize and secrete hydrolytic enzymes that break down reserves in the starchy endosperm. Unlike the cells of the starchy endosperm, aleurone cells are viable in mature grain but undergo PCD when germination is triggered or when isolated aleurone layers or protoplasts are incubated in gibberellic acid (GA). Abscisic acid (ABA) slows down the process of aleurone cell death and isolated aleurone protoplasts can be kept alive in media containing ABA for up to 6 months. Cell death in barley aleurone occurs only after cells become highly vacuolated and is manifested in an abrupt loss of plasma membrane integrity. Aleurone cell death does not follow the apoptotic pathway found in many animal cells. The hallmarks of apoptosis, including internucleosomal DNA cleavage, plasma membrane and nuclear blebbing and formation of apoptotic bodies, are not observed in dying aleurone cells. PCD in barley aleurone cells is accompanied by the accumulation of a spectrum of nuclease and protease activities and the loss of organelles as a result of cellular autolysis.     

Effects of Alteria alternata f.sp. lycopersici toxins at different levels of tomato plant cell development

Since the host-specific toxins of Alternaria alternata f. sp. lycopersici play an important role in pathogenesis, they potentially could be applied as selective agents in in vitro selection at the cellular level for disease resistance. Prerequisite for this is that sensitivity to the Alternaria alternata f.sp. lycopersici pathotoxins is manifest at the cellular level. To gain insight into cellular effects of AAL-toxins and into the mechanisms of plant insensitivity to AAL-toxins, effects of AAL-toxins on leaves, leaf discs, roots, calli, suspension cells, minicalli and protoplasts of susceptible and resistant tomato genotypes were studied. In leaves of susceptible genotypes, toxins cause severe necrosis, while in leaves of resistant genotypes necrosis was never observed. Inhibition effects of toxins were observed at all other levels in susceptible and resistant genotypes: toxins inhibited shoot induction on leaf discs, root growth and growth of calli, suspension cells and protoplasts. This indicates a cellular site for AAL-toxins. Differences in sensitivity to AAL-toxins between susceptible and resistant genotypes were observed in leaves and roots, but were not observed during shoot induction on leaf discs, in calli, suspension cells and protoplasts. However, differences in sensitivity to AAL-toxins in roots were at least 20 times less than in leaves. Therefore insensitivity seems related to a higher level of tomato plant differentiation and is most pronounced in leaves.     

维甲酸诱导的人大肠癌细胞凋亡

本研究应用光镜、电镜技术、DNA凝胶电泳、流式细胞术及末端脱氧核苷酰转移酶原位标记(TUNEL法),观察全反式维甲酸ATRA诱导的人大肠癌CCL229细胞凋亡特征。RA诱导CCL229细胞凋亡,光、电镜下观察到凋亡小体形成等典型的形态学改变,琼脂糖凝胶电泳上呈现特征性的DNA ladder,DNA直方图上显示亚二倍体峰。10-8mol/L-105mol/L范围内,RA诱导CCL229细胞凋亡表现出时间和剂量依赖性。     

拟南芥根毛衰老死亡过程的PCD检测

根毛是植物体吸收养分的重要器官, 自然条件下根毛的寿命很短, 仅能存活2–3周, 随即脱落死亡。以模式植物拟南芥(Arabidopsis thaliana)根毛为材料, 对根毛死亡的细胞学特征进行了报道。结果发现, 根毛衰老死亡后细胞内的原生质体发生了收缩, 并在胞质中观察到凝集物的出现; 通过原位末端标记(TUNEL)检测, 发现幼根上的根毛细胞核DNA发生了片段化。上述结果表明, 拟南芥根毛的衰老死亡很可能是植物体自主调控的程序性细胞死亡(PCD)。另外, 当根毛衰老死亡后,细胞核大多会迁移到靠近根毛基部的位置, 且正常的长管状根毛发生旋转扭曲。     

Deoxyribonuclease IIalpha is required during the phagocytic phase of apoptosis and its loss causes perinatal lethality

Deoxyribonuclease IIalpha (DNase IIalpha) is one of many endonucleases implicated in DNA digestion during apoptosis. We produced mice with targeted disruption of DNase IIalpha and defined its role in apoptosis. Mice deleted for DNase IIalpha die at birth with many tissues exhibiting large DNA-containing bodies that result from engulfed but undigested cell corpses. These DNA-containing bodies are pronounced in the liver where fetal definitive erythropoiesis occurs and extruded nuclei are degraded. They are found between the digits, where apoptosis occurs, and in many other regions of the embryo. Defects in the diaphragm appear to cause death of the mice due to asphyxiation. The DNA in these bodies contains 3'-hydroxyl ends and therefore stain positive in the TUNEL assay. In addition, numerous unengulfed TUNEL-positive cells are observed throughout the embryo. Apoptotic cells are normally cleared rapidly from a tissue; hence the persistence of the DNA-containing bodies and TUNEL-positive cells identifies sites where apoptosis occurs during development. These results demonstrate that DNase IIalpha is not required for the generation of the characteristic DNA fragmentation that occurs during apoptosis but is required for degrading DNA of dying cells and this function is necessary for proper fetal development.     

The involvement of jasmonates and ethylene in Alternaria alternata f. sp. lycopersici toxin-induced tomato cell death

Previous studies have shown that an ethylene (ET)-dependent pathway is involved in the cell death signalling triggered by Alternaria alternata f. sp. lycopersici (AAL) toxin in detached tomato (Solanum lycopersicum) leaves. In this study, the role of jasmonic acid (JA) signalling in programmed cell death (PCD) induced by AAL toxin was analysed using a 35S::prosystemin transgenic line (35S::prosys), a JA-deficient mutant spr2, and a JA-insensitive mutant jai1. The results indicated that JA biosynthesis and signalling play a positive role in the AAL toxin-induced PCD process. In addition, treatment with the exogenous ET action inhibitor silver thiosulphate (STS) greatly suppressed necrotic lesions in 35S::prosys leaves, although 35S::prosys leaflets co-treated with AAL toxin and STS still have a significant high relative conductivity. Application of 1-aminocyclopropane-1-carboxylic acid (ACC) markedly enhanced the sensitivity of spr2 and jai1 mutants to the toxin. However, compared with AAL toxin treatment alone, exogenous application of JA to the ET-insensitive mutant Never ripe (Nr) did not alter AAL toxin-induced cell death. In addition, the reduced ET-mediated gene expression in jai1 leaves was restored by co-treatment with ACC and AAL toxin. Furthermore, JA treatment restored the decreased expression of ET biosynthetic genes but not ET-responsive genes in the Nr mutant compared with the toxin treatment alone. Based on these results, it is proposed that both JA and ET promote the AAL toxin-induced cell death alone, and the JAI1 receptor-dependent JA pathway also acts upstream of ET biosynthesis in AAL toxin-triggered PCD.     

The mechanism of thioacetamide-induced apoptosis in the L37 albumin-SV40 T-antigen transgenic rat hepatocyte-derived cell line occurs without DNA fragmentation

Summary The hepatotoxicant thioacetamide (TH) has classically been used as a model to study hepatic necrosis; however, recent studies have shown that TH can also induce apoptosis. In this report we demonstrate that 2.68±0.54% of the albumin-SV40 T-antigen transgenic rat hepatocytes undergo TH-induced apoptosis, a level comparable to other in vivo models of liver apoptosis. In addition, TH could induce apoptosis and necrosis in the L37 albumin-SV40 T-antigen transgenic rat liver-derived cell line. Examination of dying L37 cells treated with 100 mM TH by electron microscopy revealed distinct morphological characteristics that could be attributed to apoptosis. Quantitation of apoptosis by FACS analysis 24 h after treatment with 100 mM TH revealed that 81.3±1.6% of the cells were undergoing apoptosis. In contrast, when L37 cells were treated with 250 mM TH, cells exhibited characteristics consistent with necrotic cell death. DNA fragmentation ladders were produced by growth factor withdrawal-induced apoptosis; however, in mM TH-induced apoptosis, DNA fragmentation ladders were not observed. Analysis of endonuclease activity in L37 cells revealed that the enzymes were not inactivated in the presence of 100 mM TH. The data presented in this report indicate that the L37 cell line could be used to study the mechanism of TH-induced apoptosis that was not mediated through a mechanism requiring DNA fragmentation.     

Processes of protoplast senescence and death in flax fibers: An ultrastructural analysis

Plant fibers represent specialized cells that perform a mechanical function. Their development includes the following phases, typical for the most plant cells: determination, extension growth, specialization, senescence, and death. Ultrastructural analysis of these cells has been carried out at the late phases of their development (senescence and dying off) using flax phloem fibers, a classical object for the analysis of sclerenchyma fiber formation. The results of the performed analysis show that flax fiber protoplasts remain viable until the end of a vegetation season. The ultrastructural analysis of flax phloem fibers has not revealed any typical apoptosis features. Gradual degradation of the cytoplasm starts during the active thickening of a secondary cell wall and occurs via the intensification of autolytic processes, causing a partial loss of cell content. The rupture of tonoplast is the final stage. The obtained data allow us to suppose that the protoplast dying off occurs during process of the senescence, and its program is similar to the cell death program realized in the xylem fibers of woody plants.