Polyamines modulate nitrate reductase activity in wheat leaves: involvement of nitric oxide.

In the present work, the effect of polyamines (PAs) on nitrate reductase (NR) activity was studied in wheat leaves exposed to exogenously added PAs while assessing the nitric oxide (NO) involvement in the regulation of the enzyme activity. A biphasic response was observed along the time of treatment using 0.1 mM of putrescine (Put), spermidine (Spd) or spermine (Spm). At 3 h, Spd and Spm significantly reduced NR activity by 29 or 35%, respectively, whereas at 6 h, the activity of the enzyme decreased by an average of 25%. At 21 h, Put increased NR activity by 63%, while Spd and Spm elevated the enzyme activity by 114%. NR activity, that was reduced by 0.1 mM Spm at 3 and 6 h, returned almost to control values when c-PTIO (an NO scavenger) was used, confirming that NO was involved in the inhibition of NR activity. Nitric oxide was also mediating the PA-increase of the enzyme activity at longer incubation times, evidenced when the raise in NR activity produced by 0.1 mM Spm at the longest incubation time returned to the value of the control in the presence of cPTIO. Neither the protein expression nor the nitrate content were modified by PAs treatments. The involvement of PAs and NO in the regulation of NR activity is discussed.

Reactive oxygen species formation and cell death in catalase-deficient tobacco leaf discs exposed to paraquat.

In the present work, the response of tobacco (Nicotiana tabaccum L.) wild-type SR1 and transgenic CAT1AS plants (with a basal reduced CAT activity) was evaluated after exposure to the herbicide paraquat (PQ). Superoxide anion (O (2) (.-) ) formation was inhibited at 3 or 21 h of exposure, but H(2)O(2) production and ion leakage increased significantly, both in SR1 or CAT1AS leaf discs. NADPH oxidase activity was constitutively 57% lower in non-treated transgenic leaves than in SR1 leaves and was greatly reduced both at 3 or 21 h of PQ treatment. Superoxide dismutase (SOD) activity was significantly reduced by PQ after 21 h, showing a decrease from 70% to 55%, whereas catalase (CAT) activity decreased an average of 50% after 3 h of treatment, and of 90% after 21 h, in SR1 and CAT1AS, respectively. Concomitantly, total CAT protein content was shown to be reduced in non-treated CAT1AS plants compared to control SR1 leaf discs at both exposure times. PQ decreased CAT expression in SR1 or CAT1AS plants at 3 and 21 h of treatment. The mechanisms underlying PQ-induced cell death were possibly not related exclusively to ROS formation and oxidative stress in tobacco wild-type or transgenic plants.

Nitric oxide inhibits nitrate reductase activity in wheat leaves.

Nitrate reductase (NR), a committed enzyme in nitrate assimilation, is involved in the generation of nitric oxide (NO) in plants. In wheat leaf segments exposed to sodium nitroprusside (SNP) or S-nitrosoglutathione (GSNO), NR activity was significantly reduced to different degrees between 3 and 21 h, whereas its activity was partially recovered when the NO scavenger cPTIO was used. At 21 h, NR activity decreased from 38% with 10 µM SNP to 91% with 500 µM SNP, respect to the C values. S-nitrosoglutathione reduced NR activity between 18% and 26% only at 3 h. When added directly to the incubation solution, NR activity was quickly and strongly inhibited more than 90% by 10 or 50 µM SNP, whereas 10 µM GSNO reduced the enzyme activity an average of 50%, at 30 min of incubation. l-NAME and d-arginine (nitric oxide synthase (NOS) inhibitors) increased NR activity by 14% and 52% respectively, at 21 h of exposure, leading us to suppose that endogenous NOS-dependent NO formation could also be modulating NR activity. NR protein expression was not affected by 10 or 100 µM SNP at 3 or 21 h of incubation, whereas nitration of tyrosines was not detected in the NR protein. Nitrates, which content increased along the time in the tissues, could be exerting a role in this regulation.

Reactive oxygen species formation and cell death in catalase-deficient tobacco leaf disks exposed to cadmium.

The physiological responses of tobacco (Nicotiana tabacum L.) to oxidative stress induced by cadmium were examined with respect to reactive oxygen species (ROS) formation, antioxidant enzymes activities, and cell death appearance in wild-type SR1 and catalase-deficient CAT1AS plants. Leaf disks treated with 100 or 500 microM CdCl(2) increased Evans blue staining and leakage of electrolytes in SR1 or CAT1AS plants, more pronouncedly in the transgenic cultivar, but without evidence of lipid peroxidation in any of the cultivars compared to controls. Cadmium significantly reduced the NADPH oxidase-dependent O (2)(-) formation in a dose dependent manner in SR1 very strongly at 500 microM (to 5% of the activity in the nontreated SR1 leaf disks). In CAT1AS, the NADPH oxidase activity was constitutively reduced at 50% with respect to that of SR1, but the magnitude of the decay was less prominent in this cultivar, reaching an average of 64% of the C at 21 h, for both Cd concentrations. Hydrogen peroxide formation was only slightly increased in SR1 or CAT1AS leaf disks at 21 h of exposure compared to the respective controls. Cd increased superoxide dismutase activity more than six times at 21 h in CAT1AS, but not in SR1 and reduced catalase activity by 59% at 21 h of treatment only in SR1 plants. Despite that catalase expression was constitutively lower in CATAS1 compared to SR1 nontreated leaf disks, 500 microM CdCl(2) almost doubled it only in CAT1AS at 21 h. The mechanisms underlying Cd-induced cell death were possibly not related exclusively to ROS formation or detoxification in tobacco SR1 or CAT1AS plants.