Study of intra and inter population variability of common ragweed (Ambrosia artemisiifolia L.) in relation to Amb a 1 isoforms and their allergenicity

The alien plant Ambrosia artemisiifolia L. (common ragweed) is one of the main allergenic species in Europe and especially in Northern Italy, where it has become the second cause of respiratory allergy in the last two decades. In a context of climate changing, it is very important to understand what factors the allergenicity of pollen, which is primarily determined by the presence of major allergens, depends on. Specifically it should be very interesting to define if allergenicity is dependent on environmental conditions, acting as non-heritable epigenetic factors, or on heritable elements. The objective of this thesis was to study the allergenicity variation among pollen samples from different ragweed plants/populations and to identify the mechanisms and factors contributing to the variability. To this aim, seeds from ragweed Canadian, French and Italian populations, were collected and used to grow plants both “in standard natural conditions”, where the temperature (T), humidity (H) and light (L) changed during plant development, and in “controlled conditions” where environmental parameters (T, H, L) were maintained constant all the plant vital cycle long. The two applied grow conditions were applied to identify and understand the influence of heritable and non-heritable factors (epigenetic environmental factors) on pollen allergenicity. The mechanisms underlying the change in allergenicity were investigated by studying the qualitative and quantitative variations of the isoforms of the major ragweed allergen Amb a 1 with proteomic techniques. Before this experiment, ragweed populations were genetically characterized to define the origin of Italian populations and to confirm that the genetic variability is very high within populations but low among them. The results of these analyses indicated that ragweed Italian populations have a North America origin as the French ones and that all the populations investigated show, as expected, a high genetic variability among individuals and low among them. The allergenicity of pollen was also variable among individuals and not significantly different among populations. The plant growth conditions determined the differences in the variability of allergenicity. Plants grown under controlled conditions produced pollen with similar allergenicity¬¬, while plants grown in standard conditions produced pollen with much more variable allergenicity. Electrophoresis and immunoblot analyses indicated that the variability in pollen allergenicity was mainly related to the pollen content of Amb a 1 allergen and not to a different expression and reactivity of its single principal isoforms. In conclusion, on the whole the results suggests that environmental factors (L, T, H) are responsible for the strong differences in pollen allergenicity and that these differences are not ascribed to differences in expression/reactivity of individual isoforms but to a quantitative differences involving all the considered isoforms.