Pollution from heavy metals represents one of the most important threats to crops. Among these, Nickel (Ni) represents a dangerous element, strictly related to anthropic activity and easily accumulated in plants. In this study, effects of high levels (1 mM) of Ni2+ were investigated in barley (Hordeum vulgare L. cv. Nure) grown hydroponically, inducing a severe reduction in plant growth, as well as genotoxic damage. Moreover, stress affects photosynthesis, inducing a decrease in Fv/Fm and ΦPSII and an increase in D1 protein and RuBisCO (RbcL) abundance to compensate for the loss of photosynthetic efficiency. Changes were observed in carbon metabolism, with increases in phosphofructokinase, glyceraldehyde-3P dehydrogenase-NAD+, and pyruvate kinase expression confirmed by increased proteins and activities. Notably, there was an evident rise in PEP carboxylase activity, presence, and expression. This increase boosts the TCA cycle (increased fumarase) and supports photorespiration. Evident rises were observed also for glucose-6P dehydrogenase activity and presence. Ni2+ stress induced an evident increase in enzymes involved in nitrogen metabolism: particularly, the chloroplastic GS2/Fd-GOGAT cycle and N assimilation through the cytosolic glutamate dehydrogenase reaction were enhanced. These results design a specific stress-responsive metabolism by diverting the synthesis of N-compounds through alternative C/N assimilation pathways to counteract the effects of Ni2+ toxicity. This study depicts a diversion of the main C/N metabolism network towards an increase in leaf N assimilation, using carbon skeletons from dark CO2 fixation under high Ni2+ stress. These results may provide possible targets for the improvement of heavy metal tolerance in cereals.

Pollution by Heavy Metals (HMs) represents one of the most important threatens for crops. Among HMS, Nickel (Ni++) represent a dangerous element, strictly linked to anthropic activity, which is easily accumulated in plants. In this study, the effects of Ni++ stress were investigated in barley (Hordeum vulgare L. cv. Nure) grown hydroponically and exposed to 1 mM Ni++ for up to seven days. Ni++accumulated mainly in the roots, where a 70-fold increase of the IRT1 transporter, involved in Ni uptake was observed together with a 17-fold increase in the expression of the vacuolar IREG2 transporter.Ni++ stress induced both an oxidative burst and change in primary metabolism. Different stress sensors (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX) increased their activity, presence, and expression. Similarly, Ni++ stress caused a transient increase in chlorophylls and carotenoids within 1 d, and reduced photosynthetic efficiency; furthermore, an increase was observed in those enzymes involved in carbon metabolism (RuBisCO, PEPCase, fumarase) and nitrogen assimilation (nitrate reductase, Fd- and NADH-GOGAT, glutamine synthetase).These changes resulted in an increased reductant demand by plant the H. vulgare cells, and this could be satisfied by the oxidative pentose phosphate pathway (OPPP). Therefore, we investigated in detail the possible role of glucose 6P-dehydrogenase (G6PDH) in relation to Ni++ stress. Our data show that G6PDH activity could provide NADPH to support the stress response and/or alleviate those damages induced by Ni++. Specific G6PDH isoforms can play a role in heavy metal stress resilience. Namely, cytosolic G6PDH can support the diversion of primary metabolism caused by stress. Therefore, compartmented isoforms - including the heterodimeric partner P1/2-P0-G6PDH - would be able to sustain oxidative stress induced by Ni++.

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.

This study evaluates the effects of toxic metal pollution in the highly contaminated Sarno River (South Italy), by using the aquatic moss Leptodictyum riparium in bags at 3 representative sites of the river. Biological effects were assessed by metal bioaccumulation, ultrastructural changes, oxidative stress, as Reactive Oxygen Species (ROS) production and Glutathione S-transferase (GST) activity, as well as Heat Shock Proteins 70 (HSP70s) induction. The results showed that L. riparium is a valuable bioindicator for toxic metal pollution of water ecosystem, accumulating different amounts of toxic metals from the aquatic environment. Toxic metal pollution caused severe ultrastructural damage, as well as increased ROS production and induction of GST and HSP70s, in the samples exposed at the polluted sites. To assess the role and the effect of toxic metals on L. riparium, were also cultured in vitro with Cd, Cr, Cu, Fe, Ni, Pb, Zn at the same concentrations as measured at the 3 sites. Ultrastructure, ROS, GST, and HSP70s resulted severely affected by toxic metals. Based on our findings, we confirm L. riparium as a model organism in freshwater biomonitoring surveys, and GST and HSP70s as promising biomarkers of metal toxicity.

Cadmium represents one of the most toxic pollutants in plant ecosystems: at high concentrations it can cause severe effects, such as plant growth inhibition, decrease in photosynthesis and changes in plant basal metabolism. Changes in pigments’ content, RubisCO large subunit, and D1 protein indicated a severe reduction in photosynthetic efficiency. Furthermore, the decrease of nitrate reductase activity and changes in free amino acids levels show a general stress condition of nitrogen assimilation. Cadmium increased the activities of ROS scavenging enzymes; among these, ascorbate peroxidase rate was the most noticeably increased. It is worth noting that glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.64), showed changes in both activities and occurrence during cadmium stress. Interestingly, our data suggest that G6PDH would modulate redox homeostasis under metal exposure, and possibly satisfy the increased request of reductants to counteract the oxidative burst induced by cadmium. Therefore, the results suggest that APX and G6PDH may play a pivotal role to counteract the oxidative stress induced by cadmium in young barley plants.

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.

The involvement and the efficiency of the antioxidants scavenging system upon drought were examined by comparing traditional tomato landraces with respect to an industrial commercial genotype (Red Setter); for the first time, comprehensive analyses of physiological, biochemical and molecular parameters were investigated directly under real field conditions, in a typical agricultural environment of Southern Italy. The characterization of the responses upon drought evidenced peculiar changes in stomatal conductance, ascorbate peroxidase and catalase activities and expression in drought tolerant tomato landraces, with respect to the industrial genotype. An in silico analysis (promoter and co-expression study) coupled to a phylogenetic investigation of selected enzymes was performed, reinforcing the hypothesis of a basal activation of ROS scavenging machinery in the Mediterranean landraces. Thus our data suggest a constitutive increase in the expression and activities of specific enzymes involved in ROS detoxification that can play a pivotal role in the drought response shown by tomato landraces. Therefore, traditional landraces could represent an important source of useful genetic variability for the improvement of commercial varieties; their ROS detoxifying capabilities denote peculiar aspects worth being explored to better describe their specific stress tolerance.

A cDNA coding for a plastidic P2-type G6PDH isoform from poplar (Populus tremula x tremuloides) has been used to express and purify to homogeneity the mature recombinant protein with a N-terminus His-tag. The study of the kinetic properties of the recombinant enzyme showed an in vitro redox sensing modulation exerted by reduced DTT. The interaction with thioredoxins (TRXs) was then investigated.Five cysteine to serine variants (C145S – C175S – C183S – C195S – C242S) and a variant with a double substitution for Cys175 and Cys183 (C175S/C183S) have been generated, purified and biochemically characterized in order to investigate the specific role(s) of cysteines in terms of redox regulation and NADPH-dependent inhibition.Three cysteine residues (C145, C194, C242) are suggested to have a role in controlling the NADP+ access to the active site, and in stabilizing the NADPH regulatory binding site.Our results also indicate that the regulatory disulfide involves residues Cys175 and Cys183 in a position similar to those of chloroplastic P1-G6PDHs, but the modulation is exerted primarily by TRX m-type, in contrast to P1-G6PDH, which is regulated by TRX f.This unexpected specificity indicates differences in the mechanism of regulation, and redox sensing of plastidic P2-G6PDH compared to chloroplastic P1-G6PDH in higher plants.

The present study was undertaken to investigate the expression, occurrence and activity of glucose 6 phosphate dehydrogenase (G6PDH – 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 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.