Defense/stress responses activated by chitosan in sycamore cultured cells.

Chitosan (CHT) is a natural, non-toxic, and inexpensive compound obtained by partial alkaline deacetylation of chitin, the main component of the exoskeleton of crustaceans and other arthropods. The unique physiological and biological properties of CHT make this polymer useful for a wide range of industries. In agriculture, CHT is used to control numerous pre- and postharvest diseases on various horticultural commodities. In recent years, much attention has been devoted to CHT as an elicitor of defense responses in plants, which include raising of cytosolic Ca(2+), activation of MAP kinases, callose apposition, oxidative burst, hypersensitive response, synthesis of abscisic acid, jasmonate, phytoalexins, and pathogenesis-related proteins. In this work, we investigated the effects of different CHT concentrations on some defense/stress responses of sycamore (Acer pseudoplatanus L.) cultured cells. CHT induced accumulation of dead cells, and of cells with fragmented DNA, production of H(2)O(2) and nitric oxide, release of cytochrome c from the mitochondrion, accumulation of regulative 14-3-3 proteins in the cytosol and of HSP70 molecular chaperone binding protein in the endoplasmic reticulum, accompanied by marked modifications in the architecture of this cell organelle.

Ethylene is involved in stress responses induced by fusicoccin in sycamore cultured cells.

The phytohormone ethylene is involved in many physiological and developmental processes of plants, as well as in stress responses and in the development of disease resistance. Fusicoccin (FC) is a well-known phytotoxin, that in sycamore (Acer pseudoplatanus L.) cultured cells, induces a set of stress responses, including synthesis of ethylene. In this study, we investigated the possible involvement of ethylene in the FC-induced stress responses of sycamore cells by means of Co(2+), a well-known specific inhibitor of ethylene biosynthesis. Co(2+) inhibited the accumulation of dead cells in the culture, the production of nitric oxide (NO) and of the molecular chaperone Binding Protein (BiP) in the endoplasmic reticulum induced by FC. By contrast, Co(2+) was ineffective on the FC-induced accumulation of cells with fragmented DNA, production of H(2)O(2) and release of cytochrome c from the mitochondrion, and only partially reduced the accumulation of regulative 14-3-3 proteins in the cytosol. In addition, we compared the effect of FC on the above parameters with that of the ethylene-releasing compound ethephon (2-chloroethane phosphonic acid). The results suggest that ethylene is involved in several stress responses induced by FC in sycamore cells, including a form of cell death that does not show apoptotic features and possibly involves NO as a signaling molecule.

Effect of heat stress on actin cytoskeleton and endoplasmic reticulum of tobacco BY-2 cultured cells and its inhibition by Co2+.

Temperature stress such as heat, cold, or freezing is a principal cause for yield reduction in crops. In particular, heat stress is very common and dangerous for plants since this stress can impact several plant and cellular functions. In spite of their role in sensing local stress and in controlling fundamental processes including PCD, the responses of cellular structures and organelles to heat stress are poorly investigated. In this work, we investigated the possible changes induced by mild heat stress, medium heat stress, and heat shock (HS; 5 min at 35 degrees C, 45 degrees C, or 50 degrees C, respectively) on actin cytoskeleton and endoplasmic reticulum (ER) of tobacco BY-2 cultured cells. While mild and medium heat stresses are ineffective, HS induces depolymerization of actin microfilaments and changes in ER morphology accompanied by accumulation of the HSP70 binding protein (BiP). These effects of HS are prevented by the inhibitor of ethylene production Co(2+). While the analyzed cell structures do not seem to be involved in the establishment of mild and medium heat stresses at least in this experimental system, the strong modifications induced by the treatment at 50 degrees C suggest that actin cytoskeleton and ER may be involved in the responses to HS. Besides, the inhibiting effect of Co(2+) suggests a role for ethylene as a regulative molecule in the responses to HS here observed.

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.

Fusicoccin affects cytochrome c leakage and cytosolic 14-3-3 accumulation independent of H-ATPase activation.

Fusicoccin (FC) is a well known toxin acting as a 14-3-3 protein-mediated activator of the plasma membrane H(+)-ATPase and the biochemical and physiological changes induced in the cell by this toxin have, up to now, been ascribed to the increased rate of proton extrusion by this pump leading to external acidification and cell hyperpolarization. In a recent work (Malerba M et al. 2003, Physiologia Plantarum, 119: 480-488) it was shown that, besides the previously well studied changes, FC induces a large stimulation of H(2)O(2) production, an activation of alternative respiration and a leakage of cytochrome c from mitochondria. In this article further studies on the relation between the H(2)O(2) overproduction and medium acidification are reported. The increase in the rate of H(2)O(2) accumulation is particularly evident when high concentrations of the toxin ensure a rapid acidification of the medium, but it is not obtained when the time-course of acidification is reproduced by external acid additions. The FC-dependent H(2)O(2) overproduction is strongly inhibited by inhibitors of the H(+)-ATPase activity, such as vanadate and erythrosin B, and it does not occur when the activation of the H(+)-ATPase is prevented by phenylarsine oxide (PAO), an inhibitor of the activating interaction between the enzyme and its regulative 14-3-3 protein. Interestingly, all these inhibitors only partially prevent the leakage of cytochrome c from the mitochondria. A kinetic analysis of FC-dependent changes of 14-3-3s shows that the initial increase in the plasma membrane level of these proteins, presumably due to translocation of free cytosolic forms, is followed by a remarkable increase in the level of the 14-3-3 proteins located in the cytosol. This latter change is not prevented by inhibitors of the activity or activation of the H(+)-ATPase. These results suggest that, besides the H(+)-ATPase activation, FC can induce other cell changes possibly mediated by changes of the regulative 14-3-3 proteins.