Effects of heavy metals on plastidial glucose-6- phosphate dehydrogenase: kinetic changes analysis, abundance of isoforms and determination of subcellular localization

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

Mechanism(s) of action of heavy metals to investigate the regulation of plastidic glucose-6-phosphate dehydrogenase

The regulation of recombinant plastidic glucose-6P dehydrogenase from Populus trichocarpa (PtP2-G6PDH - EC 1.1.1.49) was investigated by exposing wild type and mutagenized isoforms to heavy metals. Nickel and Cadmium caused a marked decrease in PtP2-G6PDH WT activity, suggesting their poisoning effect on plant enzymes; Lead (Pb++) was substantially ineffective. Copper (Cu++) and Zinc (Zn++) exposition resulted in strongest decrease in enzyme activity, thus suggesting a physiological competition with Magnesium, a well-known activator of G6PDH activity. Kinetic analyses confirmed a competitive inhibition by Copper, and a mixed inhibition by (Cd++). Mutagenized enzymes were differently affected by HMs: the reduction of disulfide (C175-C183) exposed the NADP+ binding sites to metals; C145 participates to NADP+ cofactor binding; C194 and C242 are proposed to play a role in the regulation of NADP+/NADPH binding. Copper (and possibly Zinc) is able to occupy competitively Magnesium (Mg++) sites and/or bind to NADP+, resulting in a reduced access of NADP+ sites on the enzyme. Hence, heavy metals could be used to describe specific roles of cysteine residues present in the primary protein sequence; these results are discussed to define the biochemical mechanism(s) of inhibition of plant plastidic G6PDH

Glucose-6-phosphate dehydrogenase plays a central role in the response of tomato (Solanum lycopersicum) plants to short and long-term drought

The present study was undertaken to investigate the expression, occurrence and activity of glucose 6 phosphate dehydrogenase (G6PDH e EC 1.1.1.49), the key-enzyme of the Oxidative Pentose Phosphate Pathway (OPPP), in tomato plants (Solanum lycopersicum cv. Red Setter) exposed to short- and long-term drought stress. For the first time, drought effects have been evaluated in plants under different growth conditions: in hydroponic laboratory system, and in greenhouse pots under controlled conditions; and in open field, in order to evaluate drought response in a representative agricultural environment. Interestingly, changes observed appear strictly associated to the induction of well known stress response mechanisms, such as the increase of proline synthesis, accumulation of chaperone Hsp70, and ascorbate peroxidase. Results show significant increase in total activity of G6PDH, and specifically in expression and occurrence of cytosolic isoform (cy-G6PDH) in plants grown in any cultivation system upon drought. Intriguingly, the results clearly suggest that abscissic acid (ABA) pathway and signaling cascade (protein phosphatase 2C e PP2C) could be strictly related to increased G6PDH expression, occurrence and activities. We hypothesized for G6PDH a specific role as one of the main reductants’ suppliers to counteract the effects of drought stress, in the light of converging evidences given by young and adult tomato plants under stress of different duration and intensity