Production of Reactive Oxygen Species, Alteration of Cytosolic Ascorbate Peroxidase, and Impairment of Mitochondrial Metabolism Are Early Events in Heat Shock-Induced Programmed Cell Death in Tobacco Bright-Yellow 2 Cells

Abstract

To gain some insight into the mechanisms by which plant cells die as a result of abiotic stress, we exposed tobacco (Nicotiana tabacum) Bright-Yellow 2 cells to heat shock and investigated cell survival as a function of time after heat shock induction. Heat treatment at 55°C triggered processes leading to programmed cell death (PCD) that was complete after 72 h. In the early phase, cells undergoing PCD showed an immediate burst in hydrogen peroxide (H 2 O 2 ) and superoxide (O 2 ⅐ -) anion production. Consistently, death was prevented by the antioxidants ascorbate (ASC) and superoxide dismutase (SOD). Actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, also prevented cell death, but with a lower efficiency. Induction of PCD resulted in gradual oxidation of endogenous ASC; this was accompanied by a decrease in both the amount and the specific activity of the cytosolic ASC peroxidase (cAPX). A reduction in cAPX gene expression was also found in the late PCD phase. Moreover, changes of cAPX kinetic properties were found in PCD cells. Production of ROS in PCD cells was accompanied by early inhibition of glucose (Glc) oxidation, with a strong impairment of mitochondrial function as shown by an increase in cellular NAD(P)H fluorescence, and by failure of mitochondria isolated from cells undergoing PCD to generate membrane potential and to oxidize succinate in a manner controlled by ADP. Thus, we propose that in the early phase of tobacco Bright-Yellow 2 cell PCD, ROS production occurs, perhaps because of damage of the cell antioxidant system, with impairment of the mitochondrial oxidative phosphorylation.In plants, programmed cell death (PCD) is responsible for removal of redundant, misplaced, or damaged cells, and, thus, contributes significantly to both development and maintenance of these multicellular organisms. Activation of PCD in plants takes place during a variety of processes including differentiation of tracheary elements (Fukuda, 2000; Yu et al., 2002) and female gametophytes (Wu and Cheun, 2000), the development of cereal endosperm and aleurone cells (Young and Gallie, 2000; Fath et al., 2002), responses to external stimuli such as the hypersensitive reaction against pathogen attacks (Lamb and Dixon, 1997; Beers and McDowell, 2001) and severe abiotic stresses (Koukalovà et al., 1997; Weaver et al., 1998; Rao and Davis, 2001). At present, however, how signaling pathways lead plant cells toward death via apoptosis and how death occurs are far from being elucidated. Mammalian and plant PCD processes share several morphological and biochemical features, including cytoplasm shrinkage, nuclear condensation, DNA laddering, expression of caspase-like proteolytic activity, and release of cytochrome c from mitochondria (Balk et al., 1999; Sun et al., 1999; Kim et al., 2003). Nonetheless, in distinction from mammals (for instance, see Atlante et al., 2003aAtlante et al., , 2003b, how plant PCD takes place remains somewhat obscure. In this context, the role of the react...

AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants

“…52 Abiotic stresses, including temperature, salinity, drought, light, ROS, and ozone, are now often regarded not as toxins, but rather as elicitors of PCD, when plant cells are chronically exposed to physiological levels of these stresses. 41,[73][74][75][76][77] However, necrosis, which represents an uncontrolled form of cell death, does occur in plant cells when they are exposed to high levels of abiotic stress that the plant cannot override with their tolerance mechanisms. ROS are pivotal mediators of PCD in plants.…”

Heat Suppresses Activation of an Auxin-Responsive Promoter in Cultured Guard Cell Protoplasts of Tree Tobacco

“…These data indicate that 9 to 12 h are required to irreversibly suppress GCP capacity for NAA-mediated BA activation. This period is much longer than those typical of heat shock experiments (Vacca et al, 2004), raising the question of whether plant cell mechanisms used to respond to extended periods of high temperature may be distinct from, or augment those used by, plant cells to respond to transitory heat shock.…”

“…Cultured plant cells can transiently accumulate ROS under heat stress (Vacca et al, 2004), and H 2 O 2 suppresses auxin-responsive promoter activation in Arabidopsis mesophyll protoplasts (Kovtun et al, 1998(Kovtun et al, , 2000. Addition of 200 mM H 2 O 2 to the culture medium did not decrease or increase significantly the mean percentages of GCP expressing GFP at 32°C (unpaired t test; n 5 3; P 5 0.25) or at 38°C (unpaired t test; n 5 3; P 5 0.77) over those of their corresponding 18-h, 13 NAA temperature controls (Table I).…”

“…Tobacco (Nicotiana tabacum) plants overexpressing NPK1, the tobacco ortholog of ANP1, are better able to withstand heat shock (Kovtun et al, 2000). While a plant's capacity to metabolize reactive oxygen species (ROS; 1 O 2 , O 2 2 , H 2 O 2 , HO d ) generally increases when it acquires thermotolerance (Suzuki and Mittler, 2006), ROS may accumulate transiently in cultured plant cells in response to heat (Vacca et al, 2004). Furthermore, GCP cultured at 38°C retain a 41-kD MAP kinase that is lost at 32°C just prior to the G1-to-S phase transition (Gushwa et al, 2003).…”

Defense induced by a bis-aryl methanone compound leads to resistance in potato against Phytophthora infestans

“…Hypersensitive cell death depends on O 2 H 2 O 2 involvement is also reported in many types of PCD induced by abiotic stress. Indeed, H 2 O 2 is involved in the PCD induced by heat shock in tobacco BY-2 cells (Locato et al 2008;Vacca et al 2004) and in the hypoxia-induced lysigenous aerenchyma formation in Arabidopsis (Mühlen-bock et al 2007). Treatment with NO generators contradictorily results in diminishing ROS effects (Noritake et al 1996;Orozco-Cárdenas and Ryan 2002) or in increasing them (Delledonne et al 2001;Murgia et al 2004).…”