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In recent decades, atmospheric pollution led to a progressive reduction of the ozone layer with a consequent increase in UV-B radiation. Despite the high adaptation of olive trees to the Mediterranean environment, the progressive increase of UV-B radiation is a risk factor for olive tree cultivation. It is therefore necessary to understand how high levels of UV-B radiation affect olive plants and to identify olive varieties which are better adapted. In this study we analyzed two Italian olive varieties subjected to chronic UV-B stress. We focused on the effects of UV-B radiation on RubisCO, in terms of quantity, enzymatic activity and isoform composition. In addition, we also analyzed changes in the activity of antioxidant enzymes (SOD, CAT, GPox) to get a comprehensive picture of the antioxidant system. We also evaluated the effects of UV-B on the enzyme sucrose synthase. The overall damage at biochemical level was also assessed by analyzing changes in Hsp70, a protein triggered under stress conditions. The results of this work indicate that the varieties (Giarraffa and Olivastra Seggianese) differ significantly in the use of specific antioxidant defense systems, as well as in the activity and isoform composition of RubisCO. Combined with a different use of sucrose synthase, the overall picture shows that Giarraffa optimized the use of GPox and opted for a targeted choice of RubisCO isoforms, in addition to managing the content of sucrose synthase, thereby saving energy during critical stress points.