Mojtaba Mohammadi

Changes in peroxidase and polyphenol oxidase activities in susceptible and resistant wheat heads inoculated with Fusarium graminearum and induced resistance

Abstract Guaiacol-peroxidase (POX) and polyphenol oxidase (PPO) activities were measured spectrophotometrically in resistant (cvs. Sumai#3 and Wang shui-bai) and susceptible (cvs. Falat and Golestan) wheat heads at flowering, milk, dough and ripening stages following the inoculation with Fusarium graminearum at anthesis. POX specific activity in resistant and susceptible wheat cultivars showed a significant increase during the milk stage as compared with the non-inoculated control plants. POX activity reached the highest level in heads of Wang shui-bai followed by those of Falat, Sumai#3 and Golestan cultivars at milk stage. The optimal pH for POX activity was 5.4. POX was inhibited by KCN. Native polyacrylamide gel revealed the presence of upto three basic (cationic) and six acidic (anionic) isozymes in wheat heads after a specific activity stain. PPO specific activity in wheat heads reached a maximum level during the milk stage and subsequently declined. This activity was three times higher in the resistant cultivars than the non-inoculated control plants. In Falat and Golestan cultivars, PPO activity level was half of those in resistant cultivars. The optimal pH for PPO was 6.4. PPO-catalyzed reaction was inhibited by ascorbic acid. Activity stain in non-denaturing polyacrylamide gel revealed the presence of one basic and six acidic isozymes in wheat heads. The susceptible Falat heads pre-treated with an autoclaved mycelial wall preparation showed induced resistance against FHB and increased activities of POX and PPO.

Superoxide anion generation in effective and ineffective soybean root nodules

Summary Superoxide anion generation was measured in soybean root nodules through the autoxidation of epinephrine, resulting in the formation of adrenochrome. Superoxide anion production was initially high in effective nodules but it rapidly declined following an increase in nitrogen fixation capacity. Superoxide anion levels sharply increased after a drop in the nitrogen fixation rate and reached their maximum rates between 35 and 42 days post-inoculation. Superoxide anion rate of formation was 2 to 4 times higher in the nodules than their respective adjacent root tissue. The rate of epinephrine autoxidation was partially dependent on EDTA and NAD(P)H. Superoxide anion production was inhibited between 80 to 90 percnt; by 100 mmol/L of HEPES, MES, MOPS, Tiron, thiourea and two units of superoxide dismutase whereas BSA, DMSO, mannitol, methionine, phenylalanine and sodium benzoate (quenchers of hydroxyl radicals) caused only a partial suppression of superoxide anion formation. On the other hand, both sodium azide and mercapto-ethanol, stimulated O2*− production.

β-1,3-Glucanase and chitinase activities in soybean root nodules

Summary β-1,3-Glucanase and chitinase specific activities were measured in soybean nodules and the adjoining root tissue during nodule ontogeny. Activities of both enzymes exhibited a transient pattern in root nodules with a maximum rate occurring following the decline in nitrogen fixation rate at 28 days post inoculation. β-1,3-Glucanase and chitinase levels were higher in the adjacent root tissue than in the nodules. β-1,3-Glucanase activity was optimal at a pH of 5.3 and an incubation temperature of 37 °C. The product formation increased linearly with time. TLC analysis of the hydrolytic products from the nodule reaction mixture revealed the presence of at least seven oligomers. ‘Western’ immunoblot analysis showed the nodule glucanases cross-reacting with antisera raised against virus-inducible tobacco ( Nicotiana tabacum L.) protein PR-1 (17 kDa), tobacco basic protein PR-2 (21 kDa), lima bean ( Phaseolus vulgaris L.) acidic protein PL1 (18 kDa), pinto bean basic protein PR-4d (21 kDa) and tobacco (36 kDa) and cowpea ( Vigna unguiculata L.) (36 kDa) PR-proteins. Chitinase was heat-labile and sensitive to mercury but was protease-resistant. A non-denaturing polyacrylamide gel activity stain revealed the presence of at least four prominent chitinolytic isoforms in nodule extract as well as in root tissue. ‘Western’ immunoblot analysis showed cross-reactivitiy of nodule chitinases with an anti-vacuolar bean leaf chitinase.

The Bt gene cry2Aa2 driven by a tissue specific ST-LS1 promoter from potato effectively controls Heliothis virescens.

Expression of the Cry2Aa2 protein was targeted specifically to the green tissues of transgenic tobacco Nicotiana tabacum cv. Xanthi plants. This deployment was achieved by using the promoter region of the gene encoding the Solanum tuberosum leaf and stem specific (ST-LS1) protein. The accumulated levels of toxin in the leaves were found to be effective in achieving 100 mortality of Heliothis virescens larvae. The levels of Cry2Aa2 expression in the leaves of these transgenic plants were up to 0.21 of the total soluble proteins. Bioassays with R1 transgenic plants indicated the inheritance of cry2Aa2 in the progeny plants. Tissue-specific expression of the Bt toxin in transgenic plants may help in controlling the potential occurrence of insect resistance by limiting the amount of toxin to only predated tissues. The results reported here validate the use of the ST-LS1 gene promoter for a targeted expression of Bt toxins in green tissues of plants.

Induced chitinase activity in resistant wheat leaves inoculated with an incompatible race of Puccinia striiformis f. sp. tritici, the causal agent of yellow rust disease.

Chitinase specific activity was measured spectrophotometrically in wheat leaf tissues during the compatible and incompatible interactions with Puccinia striiformis f. sp. tritici, the causal agent of yellow rust disease. The wheat cultivar, Federation* 4/Kavkaz, was inoculated with virulent (134E134A+) or avirulent (4EOA+) races of P. striiformis f. sp. tritici in the first leaf stage. The results showed that chitinase activity pattern was similar in both compatible and incompatible interactions up to 72 hrs after inoculation. However, the specific activity increased rapidly in the incompatible reaction thereafter. In susceptible reaction, chitinase activity gradually declined after 72 hrs post-inoculation reaching a level similar to that in the control plants two weeks after inoculation. Chitinase specific activity in resistance response was at least three times greater than that in the susceptible reaction two weeks following the inoculation. Electrophoresis of native polyacrylamide gel impregnated with 0.1% (w/v) glycol chitinas the substrate revealed the presence of eight chitinase isoforms with relative electrophoretic mobility (Rm) values ranging from 0.11 to 0.64 in the resolving gel.

Membrane lipid peroxidation, nitrogen fixation and leghemoglobin content in soybean root nodules

Summary Membrane lipids in soybean nodules may undergo oxidative degradation resulting in the loss of membrane structural integrity and physiological activities. One of the final products of lipid peroxidation is malondialdehyde (MDA), which can react with thiobarbituric acid (TBA) in vitro to form a chromogenic adduct, a Schiff base product that can be measured spectrophotometrically. MDA formation was quantified in the nodules as well as in the adjacent root tissue. Lipid peroxidation was initially high in soybean nodules induced by Bradyrhizobium japonicum, but sharply declined following an increase in both leghemoglobin content and nitrogen fixation rate. Lipid peroxidation was 2 to 4 times higher in the nodules than in their corresponding adjoining root tissue. Malondialdehyde levels in ineffective nodules were 1.5 times higher than those in effective nodules. MDA formation was also shown to occur in the ‘leghemoglobin-free’ cytosolic fraction, the ‘leghemoglobin’ fraction, and the nodule tissue pellet. Antioxidants, such as reduced ascorbic acid, glutathione, and 8-hydroxyquinoline, caused a partial suppression of lipid peroxidation, whereas ferrous sulfate, hydrogen peroxide, iron EDTA, disodium-EDTA, and β-carotene induced MDA formation. In contrast, quenchers of oxygen free radicals such as HEPES, MES, MOPS, PIPES, phenylalanine, Tiron, thiourea, sodium azide, and sodium cyanide (uncouplers of oxidative phosphorylation) caused somewhere between a 12 to 70 percnt; reduction in MDA production. TBA-reactive products were formed despite the incorporation of superoxide dismutase, proxidase, and catalase into the reaction mixture.

Induced lipoxygenases in soybean root nodules

Abstract Lipoxygenase (LOX; EC 1.13.11.12) 1 and 2&3 isozymic activities were measured spectrophotometrically in soybean nodules and their adjoining root tissue during nodule ontogeny. In effective nodules initiated by Bradyrhizobium japonicum strains 2122 and 2143, LOX1 specific activity gradually increased with nodule age. In ineffective nodules induced by strain 3122, LOX1 activity was 2–3 times less than those occurring in the corresponding effective nodules. LOX2&3 activity in effective nodules, showed a transient pattern with a peak at 35 days post-inoculation (dpi) as compared to LOX1 activity. In ineffective nodules, LOX2&3 activity peaked at 42 dpi and was half the rate observed in the effective nodules. LOX2&3 activity in the root tissues adjacent to effective nodules was extremely low and insignificant. LOX specific activities increased following the decline in nitrogen fixation capacity in root nodules at 28 dpi. The activity pattern of LOX temporally coincided with that of superoxide anion generation in nodules. LOX activity was inhibited by sodium azide, esculin, mercuric chloride and quercetin with high to low effectiveness, respectively. LOX activity was also inhibited by antioxidants such as ascorbate and reduced glutathione. Other inhibitors included superoxide dismutase, NADH, KCN and dithioerythretol. ‘Western’ immunoblot analysis revealed the cross-reactivity of 97 kDa nodule LOX with anti-soybean seed LOX antiserum. Induced LOX activity in the root nodules may suggest an early incompatibility between soybean and B. japonicum leading to nodule senescence.