Ezzedine El Ferjani

Cadmium and zinc induction of lipid peroxidation and effects on antioxidant enzyme activities in bean (Phaseolus vulgaris L.)

The relationship between Cd and Zn phytotoxicities and oxidative reactions in bean plants was studied. Ten-day-old bean (Phaseolus vulgaris L. cv. Morgan) plants treated for 96 h by 5 μM Cd and 100 μM Zn, separately, showed the same reduction of growth. In response to each metal, lipid peroxidation was enhanced in all plant organs, and catalase (CAT) activity was decreased in both roots and leaves, but not in stems. However, Cd and Zn stimulated the activity of guaiacol-dependent peroxidase only in stems, where native electrophoresis revealed, at least, two new anionic isozymes. The induction of one of these iso-guaiacol peroxidase (iso-GPX) was Zn-specific. The exposure to metals did not modify the activity of ascorbate-specific peroxidase either in roots or in stems. In leaves, where the glutathione reductase (GR) activity was assayed, increases of ascorbate peroxidase (APX) and glutathione reductase (GR) activities were recorded. These results suggest that some antioxidant enzymes can be activated, notably in upper plant parts, in response to oxidative stress induced by Cd and Zn.

Response of antioxidant enzymes to excess copper in tomato (Lycopersicon esculentum, Mill.)

Abstract 15-day-old seedlings of tomato (Lycopersicon esculentum, Mill., cv. Ibiza F1) were treated, for 7 days, by addition of 50 μM Cu to the nutrient medium. Under these conditions, a decrease in growth, more pronounced in leaves and in stems than in roots, was observed, as compared to controls. Copper accumulation was markedly higher in roots as compared to shoots. In all plant parts, excess copper induced an increase in the rate of lipid peroxidation. Catalase (CAT) activity was not modified in leaves and in stems, but it was decreased in roots. The activity of ascorbate peroxidase (APX) was unaltered in roots and in stems, while it was diminished in leaves. However, guaiacol peroxidase (GPX) activity was increased only in roots and stems. Enhancement of this activity was more important in stems than in roots. Analysis of the profile of anionic isoenzymes of GPX revealed quantitative and qualitative changes in roots and stems of stressed plants. These results, together, indicate that a toxic concentration of copper (50 μM) induces oxidative stress and differential responses of antioxidant enzymes in plant parts. Activity level of GPX, but not APX nor CAT, increased in response to damage.

Changes in antioxidant and lignifying enzyme activities in sunflower roots (Helianthus annuus L.) stressed with copper excess

Treatment with 50 microM CuSO4 for five days caused significant decrease in dry-matter production and protein level of ten-day-old sunflower seedling roots. An increase of lipoperoxidation product rate was also observed. The involvement of some enzyme activities in the sunflower root defence against Cu-induced oxidative stress was studied. Copper treatment induced several changes in antioxidant enzymes. SOD (superoxide dismutase, EC 1.15.1.1) activity was reduced but CAT (catalase, EC 1.11.1.6) and GPX (guaiacol peroxidase, EC 1.11.1.7) activities were significantly enhanced. The lignifying peroxidase activities, assayed using coniferyl alcohol and syringaldazine, were also stimulated. Analysis by native gel electrophoresis of syringaldazine peroxidase activity showed the stimulation of an isoform (A2) and the induction of another one (A1) under cupric stress conditions. On the other hand, the activity of PAL (phenylalanine ammonia lyase, EC 4.3.1.5), which plays an important role in plant defence, was also activated. The possible mechanisms by which Cu-induced growth delay and changes in enzymatic activities involved in plant defence processes are discussed.

Expression of stress-related genes in tomato plants exposed to arsenic and chromium in nutrient solution

The molecular responses of hydroponically cultivated tomato plants to As(V) or Cr(VI) were assessed by transcript accumulation analysis of genes coding for products potentially involved in heavy metal tolerance. A quantitative real-time PCR experiment was performed with Hsp90-1, MT2- and GR1-like protein genes using RNA isolated from tomato roots or shoots treated for 24h with As(V) or Cr(VI) at concentrations ranging from 80 to 640 microM. Both transient metallic treatments induced Hsp90-1 transcript accumulation in tomato plants. MT2- and GR1-like transcripts accumulated in tomato roots treated with As(V) but were only slightly affected by Cr(VI) treatment. Tomatoes showed phenotypic symptoms to heavy metal toxicity when plants were exposed to Cr(VI) but not As(V). Plant lethality was observed at 1280 microM Cr(VI), indicating that tomatoes were more tolerant to As than Cr stress under the experimental conditions used here.

Respiratory metabolism in the embryonic axis of germinating pea seed exposed to cadmium.

Seeds of pea (Pisum sativum L.) were germinated for 5d by soaking in distilled water or 5mM cadmium nitrate. The relationships among cadmium stress, germination rate, changes in respiratory enzyme activities and carbohydrates mobilization were studied. Two cell fractions were obtained from embryonic axis: (1) mitochondria, used to determine enzyme activities of citric acid cycle and electron transport chain, and (2) soluble, to measure some enzyme activities involved in fermentation and pentose phosphate pathway. Activities of malate- and succinate-dehydrogenases (MDH, SDH) and NADH- and succinate-cytochrome c reductases (NCCR, SCCR) were rapidly inhibited, while cytochrome c oxidase (CCO) was unaltered by cadmium treatment. However, this stimulated the NADPH-generating enzyme activities of the pentose phosphate pathway, glucose-6-phosphate- and 6-phosphogluconate-dehydrogenases (G6PDH, 6PGDH), as well as enzyme activity of fermentation, alcohol dehydrogenase (ADH), with concomitant inhibition in the capacity of enzyme inactivator (INADH). Moreover, Cd restricted carbohydrate mobilization in the embryonic axis. Almost no glucose and less than 7% of control fructose and total soluble sugars were available in the embryo tissues after 5d of exposure to cadmium. Cotyledonary invertase isoenzyme activity was also inhibited by Cd. The results indicate that cadmium induces disorder in the resumption of respiration in germinating pea seeds. The contribution of Cd-stimulated alternative metabolic pathways to compensate for the failure in mitochondrial respiration is discussed in relation to the delay in seed germination and embryonic axis growth.

Cadmium impairs mineral and carbohydrate mobilization during the germination of bean seeds.

The germination rate, mineral (Ca, Fe, K, Mn) and carbohydrate (starch, soluble sugars, sucrose, glucose, fructose) contents and hydrolase activities in cotyledons and embryonic axes of bean seeds subjected to cadmium stress were investigated. Compared to the control, Cd caused a reduction in germination percent, embryo growth and in distribution of biomass, mineral and sugars between cotyledon and embryonic axis and inhibited the activities of alpha-amylase and invertases: soluble acid (INV-AS), soluble neutral (INV-NS), cell wall bound acid (INV-AW). Moreover, the solute leakage into the germination medium was also used as bioindicator parameter to evaluate the toxicity of cadmium accumulation, which increased in different tissues of germinating seeds in the duration of treatment and provoked nutrient loss and, thereby, electrical conductivity enhancement in the imbibition medium. This was correlated with an impairment of membrane integrity, as evidenced by high malondialdehyde (MDA) content and lipoxygenase (LOX) activity in Cd-poisoned embryo. The contribution of solute loss at the expense of growing embryonic axis to failure in reserve mobilization after Cd exposure is emphasized in association with the delay in seed germination.

Role of the ubiquitin-proteasome pathway and some peptidases during seed germination and copper stress in bean cotyledons.

The role of the ubiquitin (Ub)-proteasome pathway and some endo- and aminopeptidases (EPs and APs, respectively) was studied in cotyledons of germinating bean seeds (Phaseolus vulgaris L.). The Ub system appeared to be important both in the early (3 days) and late (9 days) phases of germination. In the presence of copper, an increase in protein carbonylation and a decrease in reduced -SH pool occurred, indicating protein damage. This was associated with an enhancement in accumulation of malondialdehyde, a major product of lipid peroxidation, and an increase in content of hydrogen peroxide (H2O2), showing oxidative stress generation. Moreover, copper induced inactivation of the Ub-proteasome (EC 3.4.25) pathway and inhibition of leucine and proline aminopeptidase activities (EC 3.4.11.1 and EC 3.4.11.5, respectively), thus limiting their role in modulating essential metabolic processes, such as the removal of regulatory and oxidatively-damaged proteins. By contrast, total trypsin and chymotrypsin-like activities (EC 3.4.21.4 and EC 3.4.21.1, respectively) increased after copper exposure, in parallel with a decrease in their inhibitor capacities (i.e. trypsin inhibitor and chymotrypsin inhibitor activity), suggesting that these endoproteases are part of the protective mechanisms against copper stress.

Copper affects the cotyledonary carbohydrate status during the germination of bean seed.

Seeds of bean (Phaseolus vulgaris L.) were germinated by soaking in distilled water or copper chloride solution. The relationships among copper excess treatment, germination rate, dry weight, sugar contents, and carbohydrase activities in cotyledon were investigated. Heavy metal stress provoked a diminution in germination rate and biomass mobilization, as compared with the control. A drastic disorder in soluble sugars export, especially glucose and fructose liberation, was also imposed after exposure to excess copper. This restricted the starch and sucrose breakdown in reserve tissue, as evidenced by the inhibition in the activities of α-amylase and invertase isoenzymes (soluble acid, soluble neutral, cell wall-bound acid).

Cadmium induced mitochondrial redox changes in germinating pea seed

Mitochondria play an essential role in producing the energy required for seedling growth following imbibition. Heavy metals, such as cadmium impair mitochondrial functioning in part by altering redox regulation. The activities of two protein redox systems present in mitochondria, thioredoxin (Trx) and glutaredoxin (Grx), were analysed in the cotyledons and embryo of pea (Pisum sativum L.) germinating seeds exposed to toxic Cd concentration. Compared to controls, Cd-treated germinating seeds showed a decrease in total soluble protein content, but an increase in –SH content. Under Cd stress conditions, Grx and glutathione reductase (GR) activities as well as glutathione (GSH) concentrations decreased both in cotyledons and the embryo. Similar results were obtained with the Trx system: Trx and NADPH-dependent thioredoxin reductase (NTR) activities were not stimulated, whereas total NAD(P) contents diminished in the embryo. However, Cd enhanced the levels of all components of the Trx system in the cotyledons. On the other hand, Cd caused a significant increase in oxidative stress parameters such as the redox ratio of coenzymes (oxidized to reduced forms) and NAD(P)H oxidase activities. These results indicate that Cd induces differential redox responses on different seed tissues. We suggest that neither Grx system nor Trx one may improve the redox status of mitochondrial thiols in the embryo of germinating pea seeds exposed to Cd toxicity, but in the cotyledons the contribution of Trx/NTR/NADPH can be established in despite the vulnerability of the coenzyme pools due to enzymatic oxidation.

Effect of cadmium on resumption of respiration in cotyledons of germinating pea seeds.

Pea seeds (Pisum sativum L.) were germinated by soaking in H2O or 5 mM CdCl2 during a 5-day period. Enzyme activities involved in respiratory metabolism were studied in cotyledons. Mitochondrial cytochrome c oxidase and NADH- and succinate-cytochrome c reductase activities were inhibited by cadmium treatment. The effects of Cd were performed in vivo and in vitro allowing to distinguish between the direct inhibition of the enzyme activities and the influence on the same enzymes into the cell environment. However, Cd exposure stimulated an enzyme activity of fermentation and inhibited the capacity of the enzyme inactivator (alcohol dehydrogenase inactivator). Moreover, the enzyme activities of NAD(P)H-recycling dehydrogenases via secondary pentose phosphate pathway, glucose-6-phosphate- and 6-phosphogluconate-dehydrogenases, were enhanced in Cd-stressed seeds. These disturbances suggest that cadmium may inflict a serious injury on renewal of respiration. The findings will help clarify the overall mechanisms that underlie cadmium-mediated toxicity in germinating seeds.