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Populus tremula L. ×  Populus alba  L. ( Populus × canescens  (Aiton) Smith) — clone INRA 717-1-B4 saplings (50 cm apex to base and carrying 19 leaves on average) — were followed for 28 days. Half of the trees were grown in charcoal-filtered air while the other half were exposed to 120 ppb ozone for 11 h a day during the light period. The expanding leaf number 4 was tagged at the beginning of the experiment and finished expansion between 7 and 14 days. These leaves were harvested weekly for biochemical and proteome analyses using quantitative bidimensional electrophoresis (DiGE). Independent of the ozone treatment, all the analyses allowed a distinction between expanding and adult leaves. The results indicate that during the expansion phase (Days 0–7) the enzymatic machinery of the leaves is set up, and remains dynamically stable in the adult leaves (Days 14–28). Although ozone had no apparent effect on expanding leaves, the metabolic stability in fully expanded leaves observed in ozone-free plants was disturbed after 2 weeks of exposure and a stress-induced response became apparent.

Differential impact of chronic ozone exposure on expanding and fully expanded poplar leaves