Integrated approach to investigate molecular mechanisms in woody root response to bending

The studies presented in this PhD thesis have the general aim to contribute in the understanding the complex and almost unknown “world” of woody root biology. In particular the work has been focused on two major issues: 1) root response to mechanical stress and 2) mechanisms involved in lateral root emission from a secondary structure. Results of these studies are described in the chapter I and chapter II. Chapter I. The response of Populus nigra woody root to mechanical stress imposed by bending. To investigate the root response to mechanical stress, during the first period of the PhD project an experimental system represented by bent poplar taproots has been set up. The first results obtained by the analysis of this experimental system are reported in the paper published by Scippa et al., (2008). The data obtained showed that poplar taproot responds to mechanical stress increasing the lateral root emissions, biomass and lignin content, activating stress-responsive genes and altering the metabolic pathways. Starting from these results, in the second phase, to dissect the mechanisms involved in the woody root response to bending a temporal and spatial analysis have been carried out. In particular after modeling the forces distribution along the bent taproot, the morphological and lignin changes were analyzed together with the alteration of proteins profiles. The use of 2DE coupled to the MS/MS allowed the identification of 211 well resolved proteins which represent the first woody root proteome map. In addition all the data obtained at the different level of investigation were further verified and elaborated by multivariate statistical analysis identifying important temporal and spatial protein markers. Chapter II. Identification and characterization of an activation-tagged gene encoding an AP2/ERF protein that regulates lateral root emission. To investigate mechanisms involved in lateral root emission from a secondary structure, a suitable experimental system is required. In details, after screening of 627 independent activation-tagged transgenic lines in tissue culture, a transgenic hybrid poplar (Populus tremula X Populus alba), with dominant root phenotype, was identified. The cause of the observed phenotype was the hyperactivation (insertion of a strong 35S transcriptional enhancer) of the gene encoding for a protein of AP2/ERF family (PtaERF003). The root phenotype was increased through IAA application to the growth medium and metabolic profiling was characterized. The data obtained address the use of the transgene as model to further identify the molecular factors controlling lateral root emission from the secondary growth. In addition the possibility of producing trees with strongly lateral root phenotype could have significant economic and environmental benefits, including ensure of a good anchorage, slopes stabilization and reduced risk of landslides.