Paolo Crosti

Tunicamycin and Brefeldin A induce in plant cells a programmed cell death showing apoptotic features.

SummaryThe recent identification ofDAD (defender against apoptotic death) gene in plants suggests that the N-linked glycosylation of proteins could be an important control point of plant programmed cell death. In this paper we describe the effects of Tunicamycin, an inhibitor of N-linked protein glycosylation, and Brefeldin A, an inhibitor of protein trafficking from the Golgi apparatus, on sycamore (Acer pseudoplatanus L.) cell cultures. These two chemicals proved able to induce a strong acceleration of the cell death; changes in cell and nucleus morphology; an increase in DNA fragmentation, detectable by a specific immunological reaction; and the presence of oligonucleosomal-size fragments (laddering) in DNA gel electrophoresis. Moreover, Brefeldin A, but not Tunicamycin, strongly stimulated the production of hydrogen peroxide. These results indicate that also in plants chemicals interfering with the activities of endoplasmic reticulum and of Golgi apparatus strongly induce a form of programmed cell death showing apoptotic features.

Role of nitric oxide in actin depolymerization and programmed cell death induced by fusicoccin in sycamore (Acer pseudoplatanus) cultured cells

Programmed cell death (PCD) plays a vital role in plant development and is involved in defence mechanisms against biotic and abiotic stresses. Different forms of PCD have been described in plants on the basis of the cell organelle first involved. In sycamore (Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin (FC) induces cell death. However, only a fraction of the dead cells shows the typical hallmarks of animal apoptosis, including cell shrinkage, chromatin condensation, DNA fragmentation and release of cytochrome c from the mitochondrion. In this work, we show that the scavenging of nitric oxide (NO), produced in the presence of FC, by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and rutin inhibits cell death without affecting DNA fragmentation and cytochrome c release. In addition, we show that FC induces a massive depolymerization of actin filaments that is prevented by the NO scavengers. Finally, the addition of actin-depolymerizing drugs induces PCD in control cells and overcomes the inhibiting effect of cPTIO on FC-induced cell death. Vice versa, the addition of actin-stabilizing drugs to FC-treated cells partially inhibits the phytotoxin-induced PCD. These results suggest that besides an apoptotic-like form of PCD involving the release of cytochrome c, FC induces at least another form of cell death, likely mediated by NO and independent of cytochrome c release, and they make it tempting to speculate that changes in actin cytoskeleton are involved in this form of PCD.

Activation of Ethylene Production in Acer pseudoplatanus L.Cultured Cells by Fusicoccin

Summary Ethylene production by suspension cultures of sycamore cells (Acer pseudoplatanus L.) is regulated by2,4-Dichlorophenoxyacetic acid (2,4-D) and by the age of the culture. It is not stimulated by applied 1-aminocyclopropane-1-carboxylic acid (ACC). Fusicoccin enhanced the rate of ethylene formation, in addition to the stimulation of proton extrusion. The effects of fusicoccin and 2,4-D were additive. The fusicoccin dependent ethylene production was not modified by addition of ACC and, as the basal production, was strongly inhibited by aminoethoxyvinylglycine (AVG) and by cobaltous ions. Moreover it occurred without a significant increase of 1-aminocyclopropane-1-carboxylic acid per cell. The stimulation by fusicoccin could be already noticed at the first hour of treatment and reached its maximum at the third hour. The effect of fusicoccin was not prevented by cycloheximide (CHE). ≪In vitro≫ measures of ACC oxidase activity of extracts from control or fusicoccin treated cells indicated that the level of this enzyme in the cells was not changed. The data suggest that in our cultures ACC oxidase is the limiting step of ethylene production and that fusicoccin increases this activity without ≪de novo≫ synthesis of ACC oxidase.

Comparison between the effects of fusicoccin, Tunicamycin, and Brefeldin A on programmed cell death of cultured sycamore (Acer pseudoplatanus L.) cells

Summary.Programmed cell death occurs in plants during several developmental processes and during the expression of resistance to pathogen attack (i.e., the hypersensitive response). An unsolved question of plant programmed cell death is whether a unique signaling pathway or different, possibly convergent pathways exist. This problem was addressed in cultured sycamore (Acer pseudoplatanus L.) cells by comparing the effects of fusicoccin, Tunicamycin and Brefeldin A, inducers of programmed cell death with well-defined molecular and cellular targets, on some of the parameters involved in the regulation of this process. In addition to cell death, the inducers are able to stimulate the production of H2O2, the leakage of cytochrome c from mitochondria, the accumulation of cytosolic 14-3-3 proteins, and changes at the endoplasmic reticulum level, such as accumulation of the molecular chaperone binding protein and modifications in the organelle architecture. Interestingly, no additive effect on any of these parameters is observed when fusicoccin is administered in combination with Tunicamycin or Brefeldin A. Thus, these inducers seem to utilize the same or largely coincident pathways to induce programmed cell death and involvement of the endoplasmic reticulum, in addition to that of mitochondria, appears to be a common step.

Fusicoccin induces in plant cells a programmed cell death showing apoptotic features

Summary. Programmed cell death plays a pivotal role in several developmental processes of plants and it is involved in the response to environmental stresses and in the defense mechanisms against pathogen attack. It has not yet been defined which part of the death signalling mechanism and which molecules involved in programmed cell death are common to animals and plants. In this paper we show that fusicoccin, a well-known phytotoxin, induces a strong acceleration in the appearance of Evans Blue-stainable (dead) cells in sycamore (Acer pseudoplatanus L.) cultures. This fusicoccin-induced cell death shows aspects common to the form of animal programmed cell death termed apoptosis: i.e., cell shrinkage, changes in nucleus morphology, increase in DNA fragmentation detectable by a specific immunological reaction, and presence of oligonucleosomal-size fragments (laddering) in DNA gel electrophoresis. Since fusicoccin has a well-identified molecular target, the plasma membrane H+-ATPase, and thoroughly investigated physiological effects, this toxin appears to be a useful tool to study the transduction of death signals leading to programmed cell death in plants.

Regulation of 1-Aminocyclopropane-1-Carboxylic Acid Oxidase by the Plasmalemma Proton Pump in Acer pseudoplatanus L. Cultured Cells

Summary The involvement of the plasmalemma proton pump in the fusicoccin-dependent activation of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase) and of ethylene production in cultured Acer cells has been investigated. A good correspondence between the degree of acidification of the medium and the increase of ethylene production was observed for a wide range of stimulating fusicoccin concentrations. Vanadate and Erythrosin B, known as inhibitors of the plasmalemma proton pump, inhibited both the acidification of the medium and the stimulation of ethylene production; 0.6 M mannitol did not inhibit the «basal» production of ethylene either in hormone depleted cells or in cells re-supplied with growthpromoting 2,4-dichlorophenoxyacetic acid (2,4-D). On the contrary, mannitol practically suppressed the stimulation of ethylene production by fusicoccin but the acidification of the medium was only slightly reduced. Vanadate did not influence the cellular level of ACC. Mannitol almost doubled the level of ACC in the fusicoccin treated cells. The data are discussed in terms of regulation by the plasmalemma proton pump of an ACC oxidase presumably associated to the plasma membrane.

Cyclosporin A inhibits programmed cell death and cytochrome c release induced by fusicoccin in sycamore cells

Summary.Programmed cell death plays a vital role in normal plant development, response to environmental stresses, and defense against pathogen attack. Different types of programmed cell death occur in plants and the involvement of mitochondria is still under investigation. In sycamore (Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin induces cell death that shows apoptotic features, including chromatin condensation, DNA fragmentation, and release of cytochrome c from mitochondria. In this work, we show that cyclosporin A, an inhibitor of the permeability transition pore of animal mitochondria, inhibits the cell death, DNA fragmentation, and cytochrome c release induced by fusicoccin. In addition, we show that fusicoccin induces a change in the shape of mitochondria which is not prevented by cyclosporin A. These results suggest that the release of cytochrome c induced by fusicoccin occurs through a cyclosporin A-sensitive system that is similar to the permeability transition pore of animal mitochondria and they make it tempting to speculate that this release may be involved in the phytotoxin-induced programmed cell death of sycamore cells.

Ferricyanide Induced Ethylene Production is a Plasma Membrane Proton Pump Dependent 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Oxidase Activation

Summary In sycamore cells ( Acer pseudoplatanus L.) the stimulation of ethylene formation dependent on the trans-plasma membrane (trans-PM) reduction of applied ferricyanide [K 3 Fe(CN) 6 ] was previously shown to be independent of applied 1-Aminocyclopropane-1-carboxylic acid (ACC) and was tentatively ascribed to an activation of ACC oxidase. In this paper we report that in ferricyanide stimulated cells: I) the cellular level of ACC does not change II) the activation of ACC oxidase is not prevented by 1 mM cycloheximide (CHE) and ill) extracts from control and treated cells possess the same amount of ACC oxidase activity. These data confirm the activation of ACC oxidase and indicate that it does not require de novo protein synthesis but is an increase of catalytic efficiency, which is lost in the cell extracts. Nutritional experiments with control and iron depleted cells indicate that it is not related to an increased availability of Fe ++ in the growth medium. Ethylene production correlates well with proton extrusion in the medium. Inhibitors of the plasmalemma proton pump used at concentrations only slightly inhibiting the rate of ferricyanide reduction strongly inhibited the development of ACC oxidase activation. Finally, activation of ACC oxidase was not induced in K + depleted cells but it was only delayed if K + was resupplied before or concomitantly to the ferricyanide reduction. The data are discussed in terms of control of ACC oxidase activity by the plasmalemma proton pump.

Eukaryotic N10-formyl-H4folate:Methionyl-tRNAf transformylase: Some properties of the Euglena gracilis enzyme

Abstract The N 10 - formyl-H 4 folate:methionyl-tRNA fMet transformylase was extracted from Euglena cells, and the level of enzyme activity found to be of 0.15 pmol/min per 106 cells for autotrophic and 0.016 pmol/min per 106 cells for streptomycin-bleached cells. The enzyme has been purified by DEAE-cellulose and CM-Sephadex chromatography about 1000-fold from autotrophic cells and about 650-fold from chloroplasts obtained by the non-aqueous technique. A methionyl-tRNA synthetase with apparently high specificity for the initiator tRNAMet has also been found which allowed the estimation of enzyme activity even with unfractionated tRNAs from different sources, since only formylatable species were present in methionylated form. As judged from the initial velocity the Euglena transformylase showed the same kinetic behaviour towards Met-tRNAfMet from E. gracilis, Escherichia coli and yeast. The chloroplast enzyme showed different properties from those of the bacterial enzyme. It appears to have a partial requirement for K+, a higher molecular weight, and a lower Mg2+ optimum. Moreover, the chloroplast transformylase showed sigmoidal rather than hyperbolic kinetics respect to Met-tRNAfMet ( n H = 1.81 ; K′ = 4 · 10 −14 ) as well as N 10 - formyl-H 4 folate ( n H = 2.46 ; K′ = 5.2 · 10 −16 ), thus indicating a strong positive cooperativity for both substrates.

Growth-dependent changes of folate metabolism and biosynthesis in cultured Daucus carota cells

Abstract Metabolism of folates has been studied in cultures of Daucus carota cells. With the addition to the culture medium of natural folates as folic acid, 5-HCO-H 4 PteGlu, 5-CH 3 -H 4 -PteGlu and pteroic acid the final yield was scarcely affected but folic acid stimulated the initial rate of growth. On the contrary, the growth rate was reduced by 5-HCO-H 4 -PteGlu. Growth was severely inhibited by folate analogues such aminopterin and methotrexate and by sulfanilamide, the inhibition of the latter being easily reversed by equimolar amount of folic acid. Microbiological assays of cell folates with Lactobacillus casei were performed at different stages of growth, covering the entire culture cycle. Most folates can be measured only after treatment of the extracts with conjugase (γ-glutamyl-hydrolase, an enzyme removing glutamate from the glutamate chain of folates) thus indicating an high ratio between poly- and mono-plus oligo-glutamate forms. Radiochromatographic analyses of folates obtained by cells cultured in [ 3 H]folic acid show the presence, besides of tri- and penta-glutamates, of forms with very long chains, of unidentified length. A strong shortening of the glutamate chain length was obtained with aminopterin and methotrexate. The ratio poly/mono, as well as the cell amount of folates, varies during the culture cycle. Rate of folate accumulation reaches its maximum before that of culture growth and sharply decreases before the slowing down of cell divisions, suggesting that depression or repression of folate biosynthetic pathways precedes the changes in the trend of cell replication.