Comet assay as an early predictor tool to detect ozone enhanced sensitivity of vegetation in a free-air controlled long-term exposure

Abstract

Although ozone (O3) is a strong oxidant with serious impacts on plants, few studies have investigated the effects on DNA damage. Here we tested the Comet assay combined with the enzymatic isolation of leaf protoplasts. Two species differing in O3 sensitivity were used, i.e. the moderately tolerant Arbutus unedo and the very sensitive Populus maximowiczii Henry × berolinensis (or Oxford clone), exposed to three O3 levels (ambient [40.3 ppb], 1.5 times ambient O3 concentration, twice ambient O3 concentration) for one (A. unedo) or two (Oxford clone) growing seasons in an O3 Free Air Controlled Exposure (FACE) facility. We found that O3 has strong plant genotoxicity capacity and the negative effects increased with increasing exposure or stomatal uptake in both species. Interestingly, both ecophysiological and DNA damage were detected in the sensitive species, while no ecophysiological injury was observed in A. unedo although DNA was damaged before other symptoms occurred. Overall, DNA damage increased when the ecophysiological parameters decreased, confirming that DNA damage is a good proxy of the functional status of a plant. We conclude that the enzymatic isolation and Comet assay could be used as a predictor tool to reveal plant O3 DNA damage even in moderately O3 tolerant species, able to recover the abiotic stress.

Keywords

• Ozone genotoxicity
• O3 Free air controlled exposure
• Protoplast isolation
• Comet assay
• DNA damage
• Ecophysiology