Identification of three MAPKKKs forming a linear signaling pathway leading to programmed cell death in

ABSTRACT: BACKGROUND: The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily ancient mechanism of signal transduction found in eukaryotic cells. In plants, MAPK cascades are associated with responses to various abiotic and biotic stresses such as plant pathogens. MAPK cascades function through sequential phosphorylation: MAPK kinase kinases (MAPKKKs) phosphorylate MAPK kinases (MAPKKs), and phosphorylated MAPKKs phosphorylate MAPKs. Of these three types of kinase, the MAPKKKs exhibit the most divergence in the plant genome. Their great diversity is assumed to allow MAPKKKs to regulate many specific signaling pathways in plants despite the relatively limited number of MAPKKs and MAPKs. Although some plant MAPKKKs, including the MAPKKKalpha of Nicotiana benthamiana (NbMAPKKKalpha), are known to play crucial roles in plant defense responses, the functional relationship among MAPKKK genes is poorly understood. Here, we performed a comparative functional analysis of MAPKKKs to investigate the signaling pathway leading to the defense response. RESULTS: We cloned three novel MAPKKK genes from N. benthamiana: NbMAPKKKbeta, NbMAPKKKgamma, and NbMAPKKKepsilon2. Transient overexpression of full-length NbMAPKKKbeta or NbMAPKKKgamma or their kinase domains in N. benthamiana leaves induced hypersensitive response (HR)-like cell death associated with hydrogen peroxide production. This activity was dependent on the kinase activity of the overexpressed MAPKKK. In addition, virus-induced silencing of NbMAPKKKbeta or NbMAPKKKgamma expression significantly suppressed the induction of programmed cell death (PCD) by viral infection. Furthermore, in epistasis analysis of the functional relationships among NbMAPKKKbeta, NbMAPKKKgamma, and NbMAPKKKalpha (previously shown to be involved in plant defense responses) conducted by combining transient overexpression analysis and virus-induced gene silencing, silencing of NbMAPKKKalpha suppressed cell death induced by the overexpression of the NbMAPKKKbeta kinase domain or of NbMAPKKKgamma, but silencing of NbMAPKKKbeta failed to suppress cell death induced by the overexpression of NbMAPKKKalpha or NbMAPKKKgamma. Silencing of NbMAPKKKgamma suppressed cell death induced by the NbMAPKKKbeta kinase domain but not that induced by NbMAPKKKalpha. CONCLUSIONS: These results demonstrate that in addition to NbMAPKKKalpha, NbMAPKKKbeta and NbMAPKKKgamma also function as positive regulators of PCD. Furthermore, these three MAPKKKs form a linear signaling pathway leading to PCD; this pathway proceeds from NbMAPKKKbeta to NbMAPKKKgamma to NbMAPKKKalpha.     

Ultrastructural aspects and programmed cell death in the tapetal cells of Lathyrus undulatus Boiss

Programmed cell death (PCD) in the tapetum of Lathyrus undulatus L. was analyzed based on light, fluorescence and electron microscopy to characterize its spatial and temporal occurrence. Development and processes of PCD in secretory tapetal cells of Lathyrus undulatus L. were correlated with the sporogenous cells and pollen grains. At early stages of development the tapetal cells appeared similar to pollen mother cells, structurally. Concurrent with meiosis, tapetum expanded both tangentially and radially as vacuoles increased in size. Tapetal cells most fully developed at young microspore stage. However, tapetum underwent substantial changes in cell organization including nucleus morphology monitored by DAPI. The TUNEL staining confirmed the occurrence of intra-nucleosomal DNA cleavage. In addition to nuclear degeneration which is the first hallmark of PCD other diagnostic features were observed at vacuolated microspore stage intensely; such as chromatin condensation at the periphery of the nucleus, nuclear membrane degeneration, chromatin release to the cytoplasm, vacuole collapse according to tonoplast rupture, shrinkage of the cytoplasm, the increase and enlargement of the endoplasmic reticulum cisternae and disruption of the plasma membrane. After vacuole collapse due to possible release of hydrolytic enzymes the cell components degraded. Tapetal cells completely degenerated at bicellular pollen stage.     

Peroxyacetyl nitrate-induced oxidative and calcium signaling events leading to cell death in ozone-sensitive tobacco cell-line

It has long been concerned that some secondary air pollutants such as smog components, ozone (O₃) and peroxyacetyl nitrate (PAN), are highly phytotoxic even at low concentrations. Compared with the biology of O₃, we largely lack the information on the toxicity model for PAN at the cellular signaling levels. Here, we studied the cell-damaging impact of PAN using suspension culture of smog-sensitive tobacco variety (Bel-W3). The cells were exposed to freshly synthesized PAN and the induced cell death was assessed under microscope after staining with Evans blue. Involvement of reactive oxygen species (ROS) in PAN toxicity was suggested by PAN-dependently increased intracellular H₂O₂ and also by the cell-protective effects of ROS scavengers and related inhibitors. Calcium chelator also lowered the level of PAN-induced cell death, indicating that Ca²⁺ is also involved. Using a transgenic cell line expressing aequorin, an increase in cytosolic Ca²⁺ concentration responsive to the pulse of PAN, but sensitive to Ca²⁺ channel blockers, was recorded, indicating that Ca²⁺ channels are activated by PAN or PAN-derived signals. Above data show some similarity between the signaling mechanisms responsive to O₃ and PAN.     

Ceramide synthase-dependent ceramide generation and programmed cell death: involvement of salvage pathway in regulating postmitochondrial events

The sphingolipid ceramide has been widely implicated in the regulation of programmed cell death or apoptosis. The accumulation of ceramide has been demonstrated in a wide variety of experimental models of apoptosis and in response to a myriad of stimuli and cellular stresses. However, the detailed mechanisms of its generation and regulatory role during apoptosis are poorly understood. We sought to determine the regulation and roles of ceramide production in a model of ultraviolet light-C (UV-C)-induced programmed cell death. We found that UV-C irradiation induces the accumulation of multiple sphingolipid species including ceramide, dihydroceramide, sphingomyelin, and hexosylceramide. Late ceramide generation was also found to be regulated by Bcl-xL, Bak, and caspases. Surprisingly, inhibition of de novo synthesis using myriocin or fumonisin B1 resulted in decreased overall cellular ceramide levels basally and in response to UV-C, but only fumonisin B1 inhibited cell death, suggesting the presence of a ceramide synthase (CerS)-dependent, sphingosine-derived pool of ceramide in regulating programmed cell death. We found that this pool did not regulate the mitochondrial pathway, but it did partially regulate activation of caspase-7 and, more importantly, was necessary for late plasma membrane permeabilization. Attempting to identify the CerS responsible for this effect, we found that combined knockdown of CerS5 and CerS6 was able to decrease long-chain ceramide accumulation and plasma membrane permeabilization. These data identify a novel role for CerS and the sphingosine salvage pathway in regulating membrane permeability in the execution phase of programmed cell death.     

Ultrastructural and histological study of degenerative changes leading to black gills in grass shrimp exposed to a dithiocarbamate biocide

The presence of a carbohydrate-specific opsonin, distinguishable from hemolymph agglutinin, was demonstrated in the American lobster, Homarus americanus. Hemolymph opsonin, measured by the enhancement of hemocyte phagocytosis of sheep erythrocytes, was found to be more heat and acid labile than the agglutinin. Both opsonization and agglutination of sheep erythrocytes were inhibited by monosaccharides; however, maximal effects on opsonization were observed with d-(+)mannose, which did not affect agglutination. On the other hand, N-acetyl-d-glucosamine significantly inhibited agglutination but had little effect on opsonization. Fractionation of the molecules was accomplished by differential adsorption to Sephadex G-200 using a 0.15 m NaCl buffer. Hemolymph recovered in the column effluent was enriched for opsonic activity and devoid of agglutinin. However, both opsonin and agglutinin could be detected in the effluent when the column was equilibrated with a 0.48 m NaCl buffer. Neither agglutinin nor opsonin were able to pass an ultrafiltration membrane capable of retaining molecules greater than 3 × 10⁵ daltons.     

Frequency of headshaking in White Leghorn chickens in response to hormonal and environmental changes

Frequency of headshaking in chickens from two selected lines (HA and LA) known to differ in this trait was observed at various ages, during hormonal fluctuations and in different environmental surroundings. Neither hormonal changes concomitant with the initial onset of lay nor ingestion of corticosterone (which stopped egg production in 240-day-old pullets) altered headshaking frequency. When pullets reared in flocks on litter were moved at 77 days of age to individual battery cages, headshaking frequency was dramatically increased in one flock but did not change in another flock. Moving 252-day-old birds from individual cages to floor pens decreased headshaking frequency in males but not in females; returning the birds to cages did not significantly alter levels of headshaking. In older birds (252 days of age), headshaking frequency in all birds in line HA and in males of line LA were considerably greater than in LA females.     

Ultrastructural evidence for a dual function of the phloem and programmed cell death in the floral nectary of Digitalis purpurea

BACKGROUND AND AIMS: The floral nectary of Digitalis purpurea is a transitory organ with stomatal exudation of nectar. In this type of nectary, the nectar is thought to be transported to the exterior via intercellular ducts that traverse the nectariferous tissue. The latter is also traversed by a ramified system of phloem strands from which prenectar sugar is most probably unloaded. The aims of this study were to provide some of the basic information needed to evaluate the possible mechanism involved in nectar secretion and to discover the fate of the nectary. METHODS: The ultrastructure of the nectary was investigated at different stages of development by analysis of a series of ultrathin (7 x 10(-8) m) sections 7 x 10(-7) m apart from one another. Proportions of the cells typical of the nectary were documented by 3D-reconstruction and morphometry. KEY RESULTS: The phloem consisted of variably shaped sieve elements and companion cells which, as a rule, were more voluminous than the sieve elements. Direct contact between the phloem strands and intercellular ducts was observed. In contrast to the phloem, which remained structurally intact beyond the secretory phase, the nectariferous tissue exhibited degenerative changes reminiscent of programmed cell death (PCD), which started as early as the onset of secretion and progressed in a cascade-like fashion until final cell death occurred in the exhausted nectary. Hallmarks of PCD were: increased vacuolation; increase in electron opacity of individual cells; progressive incorporation of plasmatic components into the vacuole reminiscent of autophagy; degradation of plastids starting with hydrolysis of starch; deformation of the nucleus and gradual disappearance of chromatin; loss of tonoplast integrity and subsequent autolysis of the rest of cellular debris. Degeneration of the cells occurred against a background of increasing cell size. CONCLUSIONS: The cytological and anatomical evidence presented here, and calculations of the solute fluxes necessary for accumulation of starch and for the production of nectar support the view that: (a) in the foxgloves' nectary, apoplastic phloem unloading dominates, at least during exudation of nectar; (b) the obsolete nectary may be dismantled by PCD; and (c) at least the products of late nectary degradation are loaded via the apoplast into the unchanged phloem and exported to sinks elsewhere in the plant for reallocation.     

Relationship between production factors and dominance in White Leghorn hens in a study on social rank and cage design

The effects of social rank and cage shape on feeding frequency, weight gain, production rate, egg size, shell strength and overt aggressive activity were determined for White Leghorn layers housed six per cage in deep and shallow cages. Social rank significantly affected feeding frequency, production rate, egg size, and aggressive activity for birds in both cage designs. Birds ranking high in the social order fed more frequently, had higher production rates, larger eggs and delivered more aggressive head pecks than birds low in the peck order. The effect for production rate was manifested only at the sixth bird level. Significant social rank effects on weight gain, final body weight and shell strength were not observed. Birds in shallow cages fed more frequently, gained more weight and were involved in more aggressive acts with cage mates than those in deep cages.     

The lace plant: a novel model system to study plant proteases during developmental programmed cell death in vivo

Programmed cell death (PCD) plays a major role in plant development and defense throughout the plant kingdom. Within animal systems, it is well accepted that caspases play a major role in the PCD process, although no true caspases have yet to be identified in plants. Despite this, vast amounts of evidence suggest the existence of caspase-like proteases in plants. The lace plant (Aponogeton madagascariensis) forms perforations in a predictable pattern between longitudinal and transverse veins over its entire leaf surface via PCD. Due to the thin nature of the leaf, allowing for long-term live cell imaging, a perfected method for sterile culture, as well as the feasibility of pharmacological experiments, the lace plant provides an excellent model to study developmental PCD. In this review, we report the suitability of the lace plant as a novel organism to study proteases in vivo during developmentally regulated cell death.     

Use of ankle brachial pressure index to predict cardiovascular events and death: a cohort study.

OBJECTIVE: To determine whether a low ankle brachial pressure index is associated with an increased risk of cardiovascular events and death, and whether the prediction of such events could be improved by including this index. DESIGN: Cohort study. SETTING: 11 practices in Edinburgh, Scotland. SUBJECTS: 1592 men and women aged 55-74 years selected at random from the age-sex registers of 11 general practices and followed up for 5 years. MAIN OUTCOME MEASURES: Incidence of fatal and non-fatal cardiovascular events and all cause mortality. RESULTS: At baseline 90 (5.7%) of subjects had an ankle brachial pressure index < or = 0.7, 288 (18.2%) had an index < or = 0.9, and 566 (35.6%) < or = 1.0. After five years subjects with an index < or = 0.9 at baseline had an increased risk of non-fatal myocardial infarction (relative risk 1.38, 95% confidence interval 0.88 to 2.16), stroke (1.98, 1.05 to 3.77), cardiovascular death (1.85, 1.15 to 2.97), and all cause mortality (1.58, 1.14 to 2.18) after adjustment for age, sex, coronary disease, and diabetes at baseline. The ability to predict subsequent events was greatly increased by combining the index with other risk factors--for example, hypertensive smokers with normal cholesterol concentrations had a positive predictive value of 25.0%, increasing to 43.8% in subjects with a low index and decreasing to 15.6% in those with a normal index. CONCLUSION: The ankle brachial pressure index is a good predictor of subsequent cardiovascular events, and improves on predictions by conventional risk factors alone. It is simple and accurate and could be included in routine screening of cardiovascular status.     

Order of events leading to surface immunoglobulin capping: analysis of a transmembrane signal

Capping provides a rapid assay for the transduction of 1 type of membrane signal generated by the cross-linking of cell surface receptors. The order of the steps comprising this signal was determined by employing reversible inhibitors of lymphocyte surface Ig capping in a sequential incubation protocol. The results demonstrated that surface Ig cross-linking leads to capping by a linear series of discrete events. Although the steps inhibited by the calcium ionophore A23187 and the tranquilizer chlorpromazine could not be distinguished, other agents also thought to influence calcium distribution in the cell acted at different steps. The order of inhibited steps was shown to be: 1) hydrocortisone; 2) calcium ionophore or chlorpromazine; 3) cytochalasins; 4) dibucaine; 5) propranolol; 6) fluoride; 7) azide. These results suggest a model wherein cross-linked membrane Ig aggregates engage the preassembled microfilament system by means of a calcium-dependent linkage. Further calcium redistribution within the cell then leads to an energy-consuming contractile event.     

Importance of the role of calcium in programmed cell death: a review

Calcium plays an important role in physiological cell death processes such as terminal differentiation and apoptosis. Cell injury occurs when the intracellular calcium pool is disturbed, which in turn may lead to cell death. Calcium in calcium-dependent enzymes, transglutaminases, various proteases, phosphorylases and kinase, is involved in the process of cell death. Examples of such enzymes involved in cell death, and the role of calcium levels in regulation of these enzymes, are described and discussed.     

Posttranslational events leading to the assembly of photosystem II protein complex: a study using photosynthesis mutants from Chlamydomonas reinhardtii

We studied the assembly of photosystem II (PSII) in several mutants from Chlamydomonas reinhardtii which were unable to synthesize either one PSII core subunit (P6 [43 kD], D1, or D2) or one oxygen-evolving enhancer (OEE1 or OEE2) subunit. Synthesis of the PSII subunits was analyzed on electrophoretograms of cells pulse labeled with [14C]acetate. Their accumulation in thylakoid membranes was studied on immunoblots, their chlorophyll-binding ability on nondenaturating gels, their assembly by detergent fractionation, their stability by pulse-chase experiments and determination of in vitro protease sensitivity, and their localization by immunocytochemistry. In Chlamydomonas, the PSII core subunits P5 (47 kD), D1, and D2 are synthesized in a concerted manner while P6 synthesis is independent. P5 and P6 accumulate independently of each other in the stacked membranes. They bind chlorophyll soon after, or concomitantly with, their synthesis and independently of the presence of the other PSII subunits. Resistance to degradation increases step by step: beginning with assembly of P5, D1, and D2, then with binding of P6, and, finally, with binding of the OEE subunits on two independent high affinity sites (one for OEE1 and another for OEE2 to which OEE3 binds). In the absence of PSII cores, the OEE subunits accumulate independently in the thylakoid lumen and bind loosely to the membranes; OEE1 was found on stacked membranes, but OEE2 was found on either stacked or unstacked membranes depending on whether or not P6 was synthesized.     

Life and death decisions: the role of the IAPs in modulating programmed cell death

Multicellular organisms have evolved elaborate signal transduction pathways for maintaining homeostasis through the control of cell proliferation and death. The recent surge of interest in the regulation of programmed cell death has led to the rapid identification of many proteins involved in controlling and executing apoptosis. The inhibitors of apoptosis proteins (IAPs) constitute a family of highly conserved death suppressing proteins that were first identified in baculoviruses, and that has recently expanded to include at least two homologues in Drosophila melanogaster and four in rodents and humans. In this article we review the current state of IAP research. Two of the IAPs, HIAP-1 and HIAP-2, have been placed within the TNFα induced cell death pathway which involves two receptors for TNFα and multiple, overlapping signal transduction proteins. A third, X-linked gene termed XIAP, is ubiquitously expressed and appears to have a broad range of suppressor activity to a variety of apoptotic triggers. The fourth member, NAIP, has been identified as the protein product of a candidate gene for the inherited neuromuscular disorder, spinal muscular atrophy (SMA). The neuroprotective activity of NAIP in an in vivo model of cerebral ischemia has also been demonstrated. This revised version was published online in November 2006 with corrections to the Cover Date.     

The role of Apaf-1 in programmed cell death: from worm to tumor

Apoptosis or programmed cell death is an important process to eliminate unnecessary or hazardous cells. Apaf-1, a mammalian homologue of CED-4 of C. elegans, is the essential adaptor molecule in the mitochondrial pathway of apoptosis. Mice lacking Apaf-1 show accumulation of neurons in the developing central nervous system due to reduced apoptosis. Apaf-1-deficient cells are remarkably resistant to various apoptotic stimuli. Apaf-1-mediated apoptosis plays a role in the prevention of tumorigenesis. However, Apaf-1-independent cell death pathways are also indicated. In this review, we will summarize what has been learned about the role of Apaf-1 by biochemical and genetical approaches.     

Line and crossing effects in a diallel mating system with highly inbred lines of White Leghorn chickens

Summary Seven highly inbred lines of White Leghorn chickens were used in a near complete diallel mating plan during eight years. The lines originated from three different base populations selected for egg weight. Average inbreeding coefficients of parents of chicks hatching in successive years were 0.75, 0.80, 0.84, 0.86, 0.89, 0.91, 0.93 and 0.94. The composition of line, specific combining ability and reciprocal effects and their estimated values are given. These effects were estimated for age at first egg (AFE), average weight of all eggs laid to 40 weeks (EW40), body weight at 40 weeks (BW40), number of eggs to 40 weeks (EP40) and number of eggs between 41 and 60 weeks (EP60). Records of 3247 hens surviving to 40 weeks and of 3133 birds to 60 weeks could be used. Large differences between line effects could be found in all traits. They were only partly due to the preceding selection in the base populations. All specific combining effects were in the expected direction, negative for AFE and positive for EW40, BW40, EP40 and EP60. Recovery of inbreeding depression inflated these effects rather substancially. Average heterosis, defined as the relative superiority of a line combination over the mid parent value, was –11.3%, 5.8%, 7.8%, 45.1% and 35.8% for AFE, EW40, BW40, EP40 and EP60 respectively. One line showed a relative superiority in AFE of -19.3% compared to about –7.9% for all other combinations. Reciprocal or sex-linked effects were generally smaller in all traits than specific combining effects, they were considerably smaller in AFE, EP40 and EP60. General reciprocal effects could be found for several lines in one or more traits. Offspring of two lines, when used as sire lines, showed a negative correlation between reciprocal effects of egg weight and body weight.     

Physiological and biochemical events leading to vitrification of plants cultured in vitro

Water content, peroxidase activity and isoperoxidases, phenylalanine ammonia-lyase activity and phenolic content were comparatively analyzed in tissues of normal and vitreous plants cultured in vitro. The release of ethylene in flask atmospheres by normal and vitrifying plants was also measured. On the basis of the results, it is hypothesized that vitrification results from a burst of ethylene controlled by the peroxidase-IAA-oxidase system. An initiating stress (e.g. excess of cytokinins or of NH₄⁺ ions) would mediate the enhancement of the activity of soluble and membrane-bound peroxidases through a rapid modification of the phenolic level. The excess of ethylene in the atmosphere of stressed plants would retroinhibit its own biosynthesis and as a consequence decrease the activities of PAL and acidic peroxidases, thus hindering lignification processes. A parallel decrease in cellulose synthesis due to a diverted conversion of sugars to amino acids is expected (from data in the literature). Deficiency of both cellulose and lignin would allow more water uptake due to reduced wall pressure and bring about the hyperhydric malformations.