Plant ERD2-like proteins function as endoplasmic reticulum luminal protein receptors and participate in programmed cell death during innate immunity

Plant secondary metabolites, such as those derived from the phenylpropanoid pathway, have a beneficial effect on human health. Manipulation of metabolic flux in the phenylpropanoid pathway is important for achieving enhanced production of compounds such as anthocyanins, flavonoids and isoflavonoids. Here, we describe the development of a high-throughput molecular evolution approach that can be used for catalytic improvement of at least four key phenylpropanoid pathway enzymes, within the context of the metabolic pathway. This method uses yeast cells that express plant phenylpropanoid pathway enzymes, leading to formation of a colored intermediate that can be used as a readout in high-throughput screening. Here we report the identification of improved tomato peel 4-coumarate:CoA ligase variants using this approach. We found that the wild-type enzyme is strongly allosterically inhibited by naringenin, a downstream product of the pathway. Surprisingly, at least two of the improved variants are completely insensitive to feedback inhibition by naringenin. We suggest that this inhibition is exerted through a unique and previously unrecognized allosteric domain.     

LsGRP1, a class II glycine-rich protein of Lilium,confers plant resistance via mediating innate immune activation and inducing fungal programmed cell death

Defence-related LsGRP1 is a leaf-specific plant class II glycine-rich protein (GRP) involved in salicylic acid-induced systemic resistance against grey mould caused by necrotrophic Botrytis elliptica in lily (Lilium) cultivar Stargazer. The C-terminal region of LsGRP1 (LsGRP1^C) can inhibit fungal growth in vitro via a mechanism of inducing fungal apoptosis programmed cell death (PCD). In this study, the role of LsGRP1 in induced defence mechanism was investigated using LsGRP1-silenced Stargazer lily and LsGRP1-transgenic Arabidopsis thaliana. LsGRP1 silencing in lily was found to slightly inhibit plant growth and greatly increase the susceptibility to B. elliptica by suppressing callose deposition and early reactive oxygen species (ROS) accumulation. In contrast, LsGRP1-transgenic Arabidopsis showed higher resistance to Botrytis cinerea and also to Pseudomonas syringae pv. tomato DC3000 as compared to the wild type, accompanied with the enhancement of callose deposition and ROS accumulation. Additionally, LsGRP1 silencing increased plant cell death caused by B. elliptica secretion and reduced pathogen-associated molecular pattern (PAMP)-triggered defence activation in Stargazer lily. Consistently, LsGRP1 expression boosted PAMP-triggered defence responses and effector recognition-induced hypersensitive response in Arabidopsis. Moreover, fungal apoptosis PCD triggered by LsGRP1 in an LsGRP1^C-dependent manner was demonstrated by leaf infiltration with LsGRP1^C-containing recombinant proteins in Stargazer lily. Based on these results, we presume that LsGRP1 plays roles in plant defence via functioning as a pathogen-inducible switch for plant innate immune activation and acting as a fungal apoptosis PCD inducer to combat pathogen attack.     

GABAergic striatal neurons exhibit caspase-independent, mitochondrially mediated programmed cell death

GABAergic striatal neurons are compromised in basal ganglia pathologies and we analysed how insult nature determined their patterns of injury and recruitment of the intrinsic mitochondrial pathway during programmed cell death (PCD). Stressors affecting targets implicated in striatal neurodegeneration [3-morpholinylsydnoneimine (SIN-1), 3-nitropropionic acid (3-NP), NMDA, 3,5-dihydroxyphenylglycine (DHPG), and staurosporine (STS)] were compared in cultured GABAergic neurons from murine striatum by analyzing the progression of injury and its correlation with mitochondrial involvement, the redistribution of intermembrane space (IMS) proteins, and patterns of protease activation. Stressors produced PCD exhibiting slow-onset kinetics with time-dependent annexin-V labeling and eventual DNA fragmentation. IMS proteins including cytochrome c were differentially distributed, although stressors except STS produced early redistribution of apoptosis-inducing factor and Omi, suggestive of early recruitment of both caspase-dependent and caspase-independent signaling. In general, Bax mobilization to mitochondria appeared to promote IMS protein redistribution. Caspase 3 activation was prominent after STS, whereas NMDA and SIN-1 produced mainly calpain activation, and 3-NP and DHPG elicited a mixed profile of protease activation. PCD and redistribution of IMS proteins in striatal GABAergic neurons were canonical and insult-dependent, reflecting differential interplay between the caspase cascade and alternate cell death pathways.     

Identification of Pseudomonas syringae type III effectors that can suppress programmed cell death in plants and yeast

The Pseudomonas syringae pv. tomato DC3000 type III secretion system (TTSS) is required for bacterial pathogenicity on plants and elicitation of the hypersensitive response (HR), a programmed cell death (PCD) that occurs on resistant plants. Cosmid pHIR11 enables non-pathogens to elicit an HR dependent upon the TTSS and the effector HopPsyA. We used pHIR11 to determine that effectors HopPtoE, avirulence AvrPphEPto, AvrPpiB1Pto, AvrPtoB, and HopPtoF could suppress a HopPsyA-dependent HR on tobacco and Arabidopsis. Mixed inoculum and Agrobacterium-mediated transient expression experiments confirmed that suppressor action occurred within plant cells. These suppressors, with the exception of AvrPpiB1Pto, inhibited the expression of the tobacco pathogenesis-related (PR) gene PR1a. DC3000 suppressor mutants elicited an enhanced HR consistent with these mutants lacking an HR suppressor. Additionally, HopPtoG was identified as a suppressor on the basis of an enhanced HR produced by a hopPtoG mutant. Remarkably, these proteins functioned to inhibit the ability of the pro-apoptotic protein, Bax to induce PCD in plants and yeast, indicating that these effectors function as anti-PCD proteins in a trans-kingdom manner. The high proportion of effectors that suppress PCD suggests that suppressing plant immunity is one of the primary roles for DC3000 effectors and a central requirement for P. syringae pathogenesis.     

动物细胞培养过程中的细胞自然凋亡

细胞培养过程中的细胞自然凋亡是细胞受环境压力的影响而发生的现象。随着细胞自然凋亡的分子生物学和生物化学研究的深入,对以动物细胞产品生产为目的的细胞培养产业将产生极有价值的影响。采用DNA重组技术把预防细胞自然凋亡的基因导入细胞和在培基中加入具有抗细胞自然凋亡的化合物等手段已用于预防或减缓细胞培养过程中的细胞自然凋亡。这些技术将大大延长细胞达到饱和密度后的培养时间,从而使细胞培养系统的生产效率得以显著提高。     

Molecular mechanisms of programmed cell death

Programmed cell death is currently under active investigation. A recent meeting focused on the molecular machinery of programmed cell death and on its role in the pathogenesis of human diseases.     

Specific association of c‐Jun‐like immunoreactivity but not c‐Jun p39 with normal and induced programmed cell death in the chick embryo

We have examined c‐Jun protein expression by immunocytochemistry in normal and pathologically induced cell death by focusing primarily on the developing neuromuscular system of the chick embryo. Several commercially available antibodies against c‐Jun were used in combination with the TUNEL technique or propidium iodide staining for detection of cells undergoing programmed cell death (PCD). Among these, a rabbit polyclonal antibody raised against the amino acids 91‐105 mapping to the amino terminal domain of mouse c‐Jun p39 (c‐Jun/sc45) transiently immunostained the cytoplasm of dying spinal cord motoneurons at a time coincident with naturally occurring motoneuron death. Late apoptotic bodies were devoid of c‐Jun/sc45 immunoreactivity. A monoclonal antibody directed against a region corresponding to the amino acids 26‐175 of c‐Jun p39 (c‐Jun/mAB) did not specifically immunostain dying neurons, but, rather, showed nuclear immunolabeling in almost all healthy motoneurons. Experimentally induced programmed death of motoneurons by means of early limb bud ablation, axotomy, or in ovo injection of the neurotoxin β‐bungarotoxin increased the number of dying cells showing positive c‐Jun/sc45 immunoreactivity. Immunoelectron microscopy with c‐Jun/sc45 antibody showed that the signal was present in the cytoplasm without a specific association with organelles, and was also present in large lysosome‐like dense bodies inside neuritic profiles. Similar findings were obtained in different types of cells undergoing normal or experimentally induced PCD. These include dorsal root ganglion neurons, Schwann cells, muscle cells, neural tube and neural crest cells during the earliest stages of spinal cord development, and interdigital mesenchymal cells of hindlimbs. In all these cases, cells showed morphological and histochemical characteristics of apoptotic‐like PCD. By contrast, motoneurons undergoing necrotic cell death induced by the excitotoxin N‐methyl‐D ‐aspartate did not show detectable c‐Jun/sc45 immunoreactivity, although they displayed an increase in nuclear c‐Jun/mAB immunostaining. In Western blot analysis of spinal cord extracts, c‐Jun/sc45 antibody weakly detected a 39‐kD band, corresponding to c‐Jun, and more strongly detected two additional bands of 66 and 45 kD which followed developmental changes coincident with naturally occurring or experimentally stimulated apoptotic motoneuron death. By contrast, c‐Jun/mAB only recognized a single p39 band as expected for c‐Jun, and did not display changes associated with neuronal apoptosis. From these data, we conclude that the c‐Jun/sc45 antibody recognizes apoptosis‐related proteins associated with the early stages of morphological PCD in a variety of neuronal and nonneuronal cells, and that c‐Jun/sc45 is a reliable marker for a variety of developing cells undergoing programmed cell death. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 171–190, 1999     

昆虫细胞程序性死亡的研究进展

在昆虫发育和抵抗病原微生物的入侵过程中,细胞凋亡与自噬性死亡现象十分常见。昆虫细胞凋亡的研究已经取得了许多的成果,但是有关细胞自噬程序性死亡的研究还正在深入。昆虫细胞凋亡的信号通路至少有3条：一条类似于线虫细胞的凋亡信号通路,另一条类似于哺乳动物细胞的凋亡信号通路, 还有一条不依赖于胱天蛋白酶的凋亡信号通路。在昆虫的多种组织细胞中,细胞凋亡与自噬程序性死亡在信号通路上存在互串(cross talking),可以相互促进、抑制或替代。了解昆虫细胞程序性死亡对防治害虫具有一定的意义。     

BCL-2 does not control programmed cell death in the IPLB-LdFB cell line from the insect Lymantria dispar

In the insect Lymantria dispar cell line IPLB-LdFB the presence of a Bcl-2-like molecule has been demonstrated. The Western blot analysis performed on the cells incubated with 2-deoxy-D-ribose (dRib), an apoptotic inducer, revealed that, in comparison with the control, the Bcl-2 expression was unaffected. Furthermore, incubation of the insect cells with an anti-Bcl-2 polyclonal antibody inhibited the apoptotic effect induced by dRib, and provoked mitochondrial membrane depolarization without any apoptotic phenomena. Similar behaviour was observed using the K+ ionophore valinomycin. From these findings, we hypothesize that the L. dispar Bcl-2-like protein is essential for maintenance of the mitochondrial membrane potential, but not, as usually thought, for the regulation of programmed cell death.     

NO signalling in cytokinin-induced programmed cell death

Cell death can be induced by cytokinin 6-benzylaminopurine (BA) at high dosage in suspension-cultured Arabidopsis cells. Herein, we provide evidence that BA induces nitric oxide (NO) synthesis in a dose-dependent manner. A reduction in cell death can be observed when the cytokinin is supplemented with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or the nitric oxide synthase (NOS) inhibitors: 2-aminoethyl-isothiourea (AET) and N^G.-monomethyl- l -arginine ( l -NMMA), which suggests that NO is produced via a NOS and is a signalling component of this form of programmed cell death. In BA-treated cells, mitochondrial functionality is altered via inhibition of respiration. This inhibition can be prevented by addition of either cPTIO or AET implying that NO acts at the mitochondrial level.     

Developmental programmed cell death in plants

Mechanisms of plant developmental programmed cell death (PCD) have been intensively studied in recent years. Most plant developmental PCD is triggered by plant hormones, and the 'death signal' may be transduced by hormonal signaling pathways. Although there are some fundamental differences in the regulation of developmental PCD in various eukaryotes of different kingdoms, hormonal control and death signal transduction via pleiotropic signaling pathways constitute a common framework. However, plants possess a unique process of PCD execution that depends on vacuolar lytic function. Comparisons of the developmental PCD mechanisms of plants and other organisms are providing important insights into the detailed characteristics of developmental PCD in plants.     

The nuclear transporter SAD2 plays a role in calcium‐ and H2O2‐mediated cell death in Arabidopsis

In response to pathogens, plant cells exhibit a rapid increase in the intracellular calcium concentration and a burst of reactive oxygen species (ROS). The cytosolic increase in Ca²⁺ and the accumulation of ROS are critical for inducing programmed cell death (PCD), but the molecular mechanism is not fully understood. We screened an Arabidopsis mutant, sad2‐5, which harbours a T‐DNA insertion in the 18^th exon of the importin beta‐like gene, SAD2. The H₂O₂‐induced increase in the [Ca²⁺]_cyt of the sad2‐5 mutant was greater than that of the wild type, and the sad2‐5 mutant showed clear cell death phenotypes and abnormal H₂O₂ accumulation under fumonisin‐B1 (FB1) treatment. CaCl₂ could enhance the FB1‐induced cell death of the sad2‐5 mutant, whereas lanthanum chloride (LaCl₃), a broad‐spectrum calcium channel blocker, could restore the FB1‐induced PCD phenotype of sad2‐5. The sad2‐5 fbr11‐1 double mutant exhibited the same FB1‐insensitive phenotype as fbr11‐1, which plays a critical role in novo sphingolipid synthesis, indicating that SAD2 works downstream of FBR11. These results suggest the important role of nuclear transporters in calcium‐ and ROS‐mediated PCD response as well as provide an important theoretical basis for further analysis of the molecular mechanism of SAD2 function in PCD and for improvement of the resistance of crops to adverse environments.     

Biochemical characterization of programmed cell death in NGF-deprived sympathetic neurons

Young sympathetic neurons die when deprived of nerve growth factor (NGF). Under such circumstances, cell death is appropriate to the developing nervous system and requires RNA and protein synthesis. We have hypothesized the existence of an endogenous death program within neurons that is suppressed by trophic factors. The extent and timing of required changes in the synthetic events that comprise the death program are unknown. In an effort to characterize the biochemical events that mediate the death program further, we performed several experiments on embryonic rat sympathetic neurons in vitro. The death program was blocked with cycloheximide when total protein synthesis was inhibited ≥80%. When protein synthesis was inhibited within 22 ± 4 h of NGF deprivation, death was prevented in half the neurons. Hence, we define the commitment point for protein synthesis to be 22 ± 4 h. Analogously, the commitment point for RNA synthesis was 26 ± 4 h and that for NGF rescue, 24 ± 4 h. We tested the ability of a wide variety of chemicals to interfere with the death program. Most compounds tested were unable to prevent neuronal death. Some treatments, however, did save NGF-deprived neurons and were subsequently characterized. These included ultraviolet light and agents that raise intracellular concentrations of cAMP. Finally, we looked for the neuronal expression in vitro and in vivo of genes that have been associated with programmed death in other cell types, including TRPM-2/SGP-2, polyubiquitin, TGFβ-1, c-fos, and c-myc. None of these genes showed significant activation associated with neuronal death. © 1992 John Wiley & Sons, Inc.     

植物细胞程序化死亡的形态学和生理生化特征

植物细胞程序化死亡(PCD)是一种由基因控制的、主动的细胞死亡过程,它在植物正常生长发育过程中起着重要作用。发生程序化死亡的植物细胞在形态、生理生化方面表现出一些共性特点和个性特点,该文对这些特点进行了综述。     

Receptor quality control in the endoplasmic reticulum for plant innate immunity

Pattern recognition receptors in eukaryotes initiate defence responses on detection of microbe‐associated molecular patterns shared by many microbe species. The Leu‐rich repeat receptor‐like kinases FLS2 and EFR recognize the bacterial epitopes flg22 and elf18, derived from flagellin and elongation factor‐Tu, respectively. We describe Arabidopsis ‘priority in sweet life’ (psl) mutants that show de‐repressed anthocyanin accumulation in the presence of elf18. EFR accumulation and signalling, but not of FLS2, are impaired in psl1, psl2, and stt3a plants. PSL1 and PSL2, respectively, encode calreticulin3 (CRT3) and UDP‐glucose:glycoprotein glycosyltransferase that act in concert with STT3A‐containing oligosaccharyltransferase complex in an N‐glycosylation pathway in the endoplasmic reticulum. However, EFR‐signalling function is impaired in weak psl1 alleles despite its normal accumulation, thereby uncoupling EFR abundance control from quality control. Furthermore, salicylic acid‐induced, but EFR‐independent defence is weakened in psl2 and stt3a plants, indicating the existence of another client protein than EFR for this immune response. Our findings suggest a critical and selective function of N‐glycosylation for different layers of plant immunity, likely through quality control of membrane‐localized regulators.     

植物细胞程序性死亡(PCD)的研究进展

细胞死亡是动、植物生长发育过程中常见的一种生命现象 ,而细胞程序性死亡 (PCD)是细胞遵循自身生命活动程序 ,并受多种因子调控的一种积极的死亡方式。近年来随着动物中PCD研究的深入 ,植物PCD亦得到相应的研究。植物细胞程序性死亡研究不仅可揭示植物衰老、死亡的内部变化规律 ,而且可为其生长发育的调控提供依据和技术。该文试对有关PCD的特点、研究意义及近年来的研究概况与方法进行简述与评价。     

Classic myrosinase‐dependent degradation of indole glucosinolate attenuates fumonisin B1‐induced programmed cell death in Arabidopsis

The mycotoxin fumonisin B1 (FB1) causes the accumulation of reactive oxygen species (ROS) which then leads to programmed cell death (PCD) in Arabidopsis. In the process of studying FB1‐induced biosynthesis of glucosinolates, we found that indole glucosinolate (IGS) is involved in attenuating FB1‐induced PCD. Treatment with FB1 elevates the expression of genes related to the biosynthesis of camalexin and IGS. Mutants deficient in aliphatic glucosinolate (AGS) or camalexin biosynthesis display similar lesions to Col‐0 upon FB1 infiltration; however, the cyp79B2 cyp79B3 double mutant, which lacks induction of both IGS and camalexin, displays more severe lesions. Based on the fact that the classic myrosinase β‐thioglucoside glucohydrolase (TGG)‐deficient double mutant tgg1 tgg2, rather than atypical myrosinase‐deficient mutant pen2‐2, is more sensitive to FB1 than Col‐0, and the elevated expression of TGG1, but not of PEN2, correlates with the decrease in IGS, we conclude that TGG‐dependent IGS hydrolysis is involved in FB1‐induced PCD. Indole‐3‐acetonitrile (IAN) and indole‐3‐carbinol (I3C), the common derivatives of IGS, were used in feeding experiments, and this rescued the severe cell death phenotype, which is associated with reduced accumulation of ROS as well as increased activity of antioxidant enzymes and ROS‐scavenging ability. Despite the involvement of indole‐3‐acetic acid (IAA) in restricting FB1‐induced PCD, feeding of IAN and I3C attenuated FB1‐induced PCD in the IAA receptor mutant tir1‐1 just as in Col‐0. Taken together, our results indicate that TGG‐catalyzed breakdown products of IGS decrease the accumulation of ROS by their antioxidant behavior, and attenuate FB1 induced PCD in an IAA‐independent way.