RESUMO
Ozone-tolerant Bel B and ozone-sensitive Bel W3 tobacco cultivars were subjected to acute ozone fumigation (200 p.p.b. for 3 h) and the subcellular localization of H2O2 was then studied. H2O2 accumulated on the cell walls and plasma membrane of both cultivars but the accumulation pattern differed greatly. H2O2 production was high in both cultivars immediately after fumigation, but, in the tolerant Bel B cultivar, after 7 h was only detected in some spongy cells adjacent to epidermal cells. Instead, in the sensitive Bel W3 cultivar, accumulation was still abundant in the cell walls of palisade, spongy and epidermal cells at this time. Significant changes in apoplastic ascorbate pool were noted in both cultivars in the first hours after fumigation. As the reduced ascorbate content remained unchanged, the marked increase in total ascorbate must have originated from the striking increase in dehydroascorbate, particularly in the ozone-sensitive Bel W3. Exposure of plants to ozone resulted in a marked transient increase in both free and conjugated salicylic acid (SA) as well as an increase in the activity of benzoic acid 2-hydroxylase which catalyses SA biosynthesis. SA induction differed greatly in the two cultivars, in that: (1) SA accumulation was far greater in the ozone-sensitive Bel W3 cv. and (2) the maximum SA peak was delayed in Bel W3 and observed only 7 h after fumigation ended. These results suggest that a high SA content, as documented in the ozone-sensitive Bel W3 cultivar, could trigger the production of ROS with subsequent SA-mediated cell-death.
RESUMO
Treatment of the ozone-sensitive tobacco (Nicotiana tabacum L. cv Bel W3) with an ozone pulse (150 nL L(-1) 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 H2O2 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 3'-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.