De Lillo, Alessia (2017) Effects of heavy metals on plastidial glucose-6- phosphate dehydrogenase: kinetic changes analysis, abundance of isoforms and determination of subcellular localization. [Tesi di dottorato]

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Heavy metals (HM) represent one of the most dangerous sources of pollution in Ecosystems. The exposition to metals induces in plants a number of responses through different mechanism, as the induction of an oxidative stress, in its turn resulting in the increase of enzymes activities involved in ROS-scavenging; among these, glucose 6P dehydrogenase (G6PDH EC 1.1.1.49) plays a pivotal role in the response to abiotic stress in plants (Esposito et al., 2005; 2015). A correlation between heavy metals and G6PDH activity has been previously demonstrated in different organisms (Slaski et al. 1996; Wei Hu et al. 2013). This project aims to analyse the effects of heavy metals on recombinant wt and cys-to-ser mutants of plastidial (P2-type)-G6PDH from Populus trichocarpa (PtP2-G6PDH), and investigate the possible changes in G6PDH, and others enzymes, activities and abundance in barley plants exposed to different heavy metals (Ni++, Cd++, Pb++, Cu++, Zn++). After incubation in vitro with different HMs, changes in activities of wt isoform and mutants of PtP2-G6PDH were observed, confirming an important role of the cysteine residues in the regulation of the PtP2-G6PDH. Furthermore, the data suggest that the inhibition by different metals of PtP2-G6PDH activity could be due to a competition between HMs and magnesium, confirming an important role of the Mg in the enzyme structure stabilization. Moreover, the experiments done on barley plants reveal changes in physiological, biochemical and bimolecular aspects, as variations in the levels and activities of several enzymes (G6PDH, catalase, NADH-GOGAT, PEPcarboxylase, fumarase, ascorbate peroxidase) indicating a correlation between heavy metals and induction of expression and activity of these basal enzymes. Interestingly, barley P2-G6PDH (HvP2-G6PDH) displays an unusually long plastidic transit peptide (>95aa), considering these sequences generally comprise less than 60aa. For this reason, we investigated the subcellular localization of HvP2-G6PDH reporter fusions in Arabidopsis protoplasts, and tobacco leaves; specifically, whether this protein may be directed to heterotrophic plastids only, or to other compartments as well. Our results reveal a localization of HvP2-G6PDH in plastids of heterotrophic cells and a possible interaction between P0-G6PDH and P2-G6PDH isoforms, possibly resulting in a sublocalization of HvP2-G6PDH into peroxisomes, in order to counteract the stress. Further studies are needed to confirm the P2-P0 interaction, and detail the sub cellular localization of the heterodimers.

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