• ozone stress
• differentially expressed genes
• programmed cell death

Tropospheric ozone (O3) represents today one of the most common and harmful air pollutants. In spite of control measures for ambient ozone containment episodic peaks are foreseen to further increase in the future climate scenarios, both in intensities and frequencies. In this perspective, an increasing number of organisms would be exposed to its strong oxidizing power, with serious consequences in terms of bio-diversity reduction, plant productivity decrease, forest decline and economical losses. Arboreal species are particularly affected in growth and productivity by ozone. These dramatic perspectives determine the need to better understand the effects induced in the most susceptible plants, and the responses that the plants deploy against this pollutant in order to adapt themselves and survive under stress conditions.
This study was aimed to contribute to better understanding of the responses induced by O3 in the trees. For these purposes, two poplar clones differentially susceptible in terms of leaf injuries (Populus deltoides x maximowiczii, Eridano clone, O3-sensitive, and Populus x euoramericana, I-214 clone, O3-tolerant) were exposed to a realistic O3 peak and the responses elicited in the leaves, the first target for O3 phytotoxic action, were compared. I focused on the morpho-anatomical leaf traits influencing the differential ozone sensitivity displayed by the two clones and also on the different responses evoked by the O3 in leaves from the two hybrids, in terms of cell death processes and differently expressed genes.
Morpho-anatomical investigations showed that the leaf anatomy in sensitive poplar hybrids (amphistomatic leaves and high stomatal density, and low density of mesophyll cell packing) may favour O3 uptake in apoplast spaces, predisposing the mesophylls to an increased oxidative damage risk.
An acute O3-fumigation had a deleterious effect only on the leaves of the sensitive clone that, within 24-48 hrs after fumigation end, displayed macroscopically detectable necrotic spots in the interveinal areas of the adaxial leaf surface, sometimes reaching the 60% of the whole leaf surface. Microscopical investigations showed that mesophyll cells appeared to be the preferential targets for O3. At precocious phases of ozone fumigation, the palisade cells displayed a marked increase in the nuclear/nucleolar functional activity (cytologically detectable as a transient augmented number of nucleoli per nucleus and as an increase in size of both nuclei and nucleoli) directly referable to ozone stress. The lesion formation was preceded at cellular level by some apoptotic hallmarks. At mid ozone treatment the externalization of phosphatidylserine residues was evidenced by using Annexin V binding assay. The cell membrane asymmetry loss was followed by a highly localized cell death process, as evidenced by the ordered distribution pattern of Evans blue stained cells. At fumigation end, in the most affected mesophyll zones, the nucleoli disappeared and the nuclei, strictly associated to degenerating cell walls, displayed an irregular shape, with highly condensed and fragmented chromatin. Clusters of TUNEL-positive cells appeared at fumigation end in mesophylls, demonstrating that DNA fragmentation events take place after ozone fumigation. Accordingly, DNA electrophoretic analyses evidenced, at ozone-fumigation end a smear of fragments, having a mean complexity of 1,4 kbp and representing a percentage superior to 40% of the total extracted DNA. In addition, vacuoles lysis, incomplete cell wall breakage and collapse, plasmolysis, intact organelles occurring until a late stage of cell degeneration, final loss of any cell content were also observed in the most injured mesophylls. Concurrently, remarkable biochemical and physiological events take place in mesophyll tissues of the sensitive leaves. A rapid accumulation of callose in the cell walls around the dying cells, as well as an increase in condensed tannins and autofluorescent compounds (polyphenols) were evidenced. A local biphasic increase in intrinsic peroxides was accompanied by an NO overproduction in the more symptomatic mesophylls, suggesting an involvement of these species in cell death induction, progression and containment processes. Significant alterations reliable to ozone stress were not evidenced in the tolerant poplar clone leaves.
In order to understand the genes differentially expressed in sensitive and tolerant poplar leaves before and after an acute O3 stress, we used a microarray containing cDNAs derived from different plant systems subjected to biotic and abiotic stresses. About the 22% of all transcripts resulted differentially regulated overtime in the two poplar clones at the chosen time-points: the majority of these belonged to cell metabolism (primary and secondary) and Disease/defence categories and resulted mainly down-regulated in the sensitive clone than in the tolerant one. At the fumigation end, during the next periods, the functional categories having the major number of differentially transcribed genes were those related to cell metabolism, signal transduction pathways. Interestingly, a significant increase in expressed genes from Disease/defence and Protein synthesis categories was evidenced after ozone stress. Interestingly, some genes regulated by ozone resulted also regulated by other stress kinds in different organisms, accordingly to the existence of conserved gene clusters involved in stress responses.
Taking into consideration all our results, I propose that an acute ozone stress may elicit programmed cell death processes in the leaves of Populus deltoides x maximowiczii, Eridano clone, having the biological significance to limit the spreading of oxidative burst and consequently to preserve the integrity of leaf tissues.
The different ozone sensitivity displayed by the two clones can be related to the different morpho-anatomical leaf characteristics that allow or prevent ozone uptake and diffusion in apoplastic spaces of mesophylls. Besides, considering the differences in the expressed transcriptome displayed by the two clones, I suggest that a more or less efficient deployment of defence mechanisms aimed to minimize the toxic effect of O3 or its by-products can explain the differences in ozone sensitivity showed by the two hybrids.