Ozone-Induced Cell Death in Tobacco Cultivar Bel W3 Plants. The Role of Programmed Cell Death in Lesion Formation

Abstract

Treatment of the ozone-sensitive tobacco (Nicotiana tabacum L. cv Bel W3) with an ozone pulse (150 nL L Ϫ1 for 5 h) induced visible injury, which manifested 48 to 72 h from onset of ozone fumigation. The "classical" ozone symptoms in tobacco cv Bel W3 plants occur as sharply defined, dot-like lesions on the adaxial side of the leaf and result from the death of groups of palisade cells. We investigated whether this reaction had the features of a hypersensitive response like that which results from the incompatible plant-pathogen interaction. We detected an oxidative burst, the result of H 2 O 2 accumulation at 12 h from the starting of fumigation. Ozone treatment induced deposition of autofluorescent compounds and callose 24 h from the start of treatment. Total phenolic content was also strongly stimulated at the 10th and 72nd h from starting fumigation, concomitant with an enhancement in phenylalanine ammonia-lyase a and phenylalanine ammonia-lyase b expression, as evaluated by reverse transcriptase-polymerase chain reaction. There was also a marked, but transient, increase in the mRNA level of pathogenesis-related-1a, a typical hypersensitive response marker. Overall, these results are evidence that ozone triggers a hypersensitive response in tobacco cv Bel W3 plants. We adopted four criteria for detecting programmed cell death in ozonated tobacco cv Bel W3 leaves: (a) early release of cytochrome c from mitochondria; (b) activation of protease; (c) DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling of DNA 3Ј-OH groups; and (d) ultrastructural changes characteristic of programmed cell death, including chromatin condensation and blebbing of plasma membrane. We, therefore, provide evidence that ozone-induced oxidative stress triggers a cell death program in tobacco cv Bel W3.Ozone (O 3 ) enters the leaf and reacts in the apoplast to produce reactive oxygen species (ROS), such as superoxide anion (⅐O 2 Ϫ ), hydrogen peroxide, hydroxyl radical (OH⅐) and singlet oxygen ( 1 O 2 ; Mudd, 1997). This triggers the plants to produce an oxidative burst by as yet unknown mechanisms (Schraudner et al., 1996). The effects of O 3 on plants are numerous and vary with the intensity and duration of exposure. Short high-peak concentrations induce cell death lesions reminiscent of lesions activated during plant-pathogen interaction (Rao et al., 2000). Among herbaceous plants, tobacco (Nicotiana tabacum) is known to be particularly sensitive to ozone and the ozone-sensitive tobacco cv Bel W3 has been widely used as biomonitor of tropospheric ozone (Heggestad, 1991). The "classical" ozone symptoms in tobacco cv Bel W3 plants occur as sharply defined dot-like lesions on the adaxial side of the leaf resulting from death of group of palisade cells (Loreto et al., 2001).Plant cell death occurs as programmed cell death (PCD) or necrosis. PCD is genetically controlled and shares some characteristic features with animal apoptosis, such as cell shrinkage, cytoplasmic condensation, chromatin condensatio...

Heterotrimeric G protein signaling in theArabidopsisunfolded protein response

“…Relative electrolyte leakage is the ratio of the fraction obtained with leaves of treated plants to that obtained with leaves from untreated control plants. Cell death was detected histochemically by trypan blue staining as described (51).…”

Increasing Tolerance to Ozone by Elevating Foliar Ascorbic Acid Confers Greater Protection against Ozone Than Increasing Avoidance

“…5B), but increased immediately following exposure to ozone and increased further, i.e. the oxidative burst previously described following ozone exposure (Schraudner et al, 1998;Wohlgemuth et al, 2002;Pasqualini et al, 2003), during the recovery period. The level of apoplastic H 2 O 2 was higher in DHAR-KD leaves and lower in DHAR-OX leaves relative to the control before and after ozone treatment, but the level increased disproportionately in DHAR-KD leaves and increased least in DHAR-OX leaves (Fig.…”

“…H 2 O 2 can also inhibit CO 2 assimilation by inhibiting several Calvin cycle enzymes (Asada, 1999). ROS can also be generated in response to exposure to pollutants, such as ozone (Grimes et al, 1983;Mudd, 1997;Schraudner et al, 1998), which can serve as signaling intermediates in guard cells to promote stomatal closure (Pei et al, 2000;Zhang et al, 2001) or, following a severe exposure, can damage cell membranes or even induce programmed cell death Overmyer et al, 2000;Rao et al, 2000;Loreto et al, 2001;Pasqualini et al, 2002Pasqualini et al, , 2003.Asc is the most abundant antioxidant in plants and serves as the major contributor to the cell redox state (Smirnoff, 2000). The role that Asc plays in responding to oxidative stress has been shown with Arabidopsis (Arabidopsis thaliana) vtc mutants that are impaired in Asc biosynthesis (Conklin et al, 1999(Conklin et al, , 2000.…”

NaJAZh Regulates a Subset of Defense Responses against Herbivores and Spontaneous Leaf Necrosis inNicotiana attenuataPlants

“…PCD is closely associated with the accumulation of ROS in plant cells during the hypersensitive response (Allan and Fluhr, 1997; Desikan et al, 1998;Houot et al, 2001;Mittler et al, 2004;Cheeseman, 2007;Quan et al, 2008), which is known to inhibit the growth of biotrophic pathogens in plants. PCD also occurs in response to wounding, ozone and UV exposure, and cold and high-light stress (Pennell and Lamb, 1997;Buckner et al, 2000;Rao et al, 2000aRao et al, , 2000bBeers and McDowell, 2001;Pasqualini et al, 2003). In addition, PCD is associated with plant growth and development; it occurs during senescence, pollen development, and vascular tissue differentiation (Wang et al, 1996;Pennell and Lamb, 1997;CalderonUrrea and Dellaporta, 1999;Buckner et al, 2000;Wu and Cheun, 2000;Lee and Chen, 2002).…”