Changes in seed water status as characterized by NMR in developing soybean seed grown under moisture stress conditions.

Changes in water status of developing seeds of Soybean (Glycine max L. Merrill.) grown under different moisture stress conditions were characterized by proton nuclear magnetic resonance (NMR)- spin-spin relaxation time (T2). A comparison of the seed development characteristics, composition and physical properties indicated that, characteristics like seed weight, seed number/ear, rate of seed filling increased with development stages but decreased with moisture stress conditions. The NMR- spin-spin relaxation (T2) component like bound water increased with seed maturation (40-50%) but decreased with moisture stress conditions (30-40%). The changes in seed water status to increasing levels of moisture stress and seed maturity indicates that moisture stress resulted in more proportion of water to bound state and intermediate state and less proportion of water in free-state. These changes are further corroborated by significant changes in protein and starch contents in seeds under high moisture stress treatments. Thus seed water status during its development is not only affected by development processes but also by moisture stress conditions. This study strongly indicated a clear moisture stress and development stage dependence of seed tissue water status in developing soybean seeds.

Pre-treatment of seeds with static magnetic field ameliorates soil water stress in seedlings of maize (Zea mays L.).

The effect of magnetic field (MF) treatments of maize (Zea mays L.) var. Ganga Safed 2 seeds on the growth, leaf water status, photosynthesis and antioxidant enzyme system under soil water stress was investigated under greenhouse conditions. The seeds were exposed to static MFs of 100 and 200 mT for 2 and 1 h, respectively. The treated seeds were sown in sand beds for seven days and transplanted in pots that were maintained at -0.03, -0.2 and -0.4 MPa soil water potentials under greenhouse conditions. MF exposure of seeds significantly enhanced all growth parameters, compared to the control seedlings. The significant increase in root parameters in seedlings from magnetically-exposed seeds resulted in maintenance of better leaf water status in terms of increase in leaf water potential, turgor potential and relative water content. Photosynthesis, stomatal conductance and chlorophyll content increased in plants from treated seeds, compared to control under irrigated and mild stress condition. Leaves from plants of magnetically-treated seeds showed decreased levels of hydrogen peroxide and antioxidant defense system enzymes (peroxidases, catalase and superoxide dismutase) under moisture stress conditions, when compared with untreated controls. Mild stress of -0.2 MPa induced a stimulating effect on functional root parameters, especially in 200 mT treated seedlings which can be exploited profitably for rain fed conditions. Our results suggested that MF treatment (100 mT for 2 h and 200 for 1 h) of maize seeds enhanced the seedling growth, leaf water status, photosynthesis rate and lowered the antioxidant defense system of seedlings under soil water stress. Thus, pre sowing static magnetic field treatment of seeds can be effectively used for improving growth under water stress.

Proton NMR transverse relaxation time and membrane stability in wheat leaves exposed to high temperature shock.

Electrolyte leakage from leaves and NMR transverse relaxation time (T2) of leaf water were used to differentiate between heat-tolerant (NIAW 845) and susceptible (HD 2428) wheat (Triticum aestivum L.) cultivars. The leaves were exposed to high temperature shock in the range 30 to 55 degrees C and the damage caused, when evaluated by the two approaches was in close agreement. The critical temperature of injury leading to loss of membrane integrity was lower (39.1 degrees C) for susceptible cultivar, compared to tolerant cultivar (44.2 degrees C). Component analyses of NMR data revealed the existence of two fractions of cellular water in leaf tissues, namely, bound and free bulk water with distinct relaxation times. A dramatic reduction in the proportion of free water and a corresponding increase in bound water was observed in response to increase in temperature. This change in proportion occurred around 38 degrees C and 43 degrees C in HD 2428 and NIAW 845 respectively. The high temperature induced irreversible damage to cellular membrane integrity led to loss of compartmentation of cellular water fractions. The tolerant cultivar maintained its membrane integrity and cell water compartmentation until a temperature of 43 degrees C and susceptible could maintain it only until 38 degrees C.

Characterisation of germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy.

Experiments were conducted to characterise the changes, especially of water status in germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy. NMR relaxation time ( T(2)) measurements showed tri-phasic or bi-phasic characteristics during different stages of hydration, depending on the seed's ability to germinate. Component analysis of T(2) data revealed the existence of only two components, bound and bulk water, in dry seeds. In contrast, both the germinating and non-germinating wheat seeds had a three-component water proton system (bound, bulk and free water) in phase I of hydration. During the lag phase (phase II) of hydration, bulk water component of non-germinating seeds disappeared completely, resulting in a two component water proton system. Nevertheless, the three component water proton system was observed in the germinating seeds in phase II. Following phase II, rapid hydration (phase III) was observed in germinating seeds only. Water protons were re-organised and there were increases in bulk and free water but decreases in bound water concomitantly. Comparison of the physical state of water in these seeds by NMR spectroscopy with that of tissue leachate conductivity measurement suggests that the seed membrane system was affected more evidently in non-germinating seeds, leading to the disorganised cell structure. The present study provides evidence that the reorganisation of physical state of water in germinating wheat seeds during hydration is essential for its subsequent event of germination.

Nitration of veratryl alcohol by lignin peroxidase and tetranitromethane.

Lignin peroxidase (LiP), from Phanerochaete chrysosporium, in the presence of H2O2 and tetranitromethane (TNM), oxidizes veratryl (3,4-dimethoxybenzyl) alcohol (VA) (I) to veratraldehyde (IV), 4,5-dimethoxy-2-nitrobenzyl alcohol (V), and 3,4-dimethoxy-nitrobenzene (VI). The formation of these products is explained by a mechanism involving the one-electron oxidation of VA by LiP to produce the corresponding cation radical, which loses a proton to generate the benzylic radical. The latter reduces TNM to generate the trinitromethane anion (VIII) and the nitrogen dioxide radical (.NO2). .NO2 couples with the VA cation radical, and the subsequent loss of a proton leads to V. Alternatively, the attack of .NO2 at C-1 of the VA cation radical, followed by aromatization and loss of formaldehyde (VII), yields VI. Isotopic labeling experiments confirm that V is generated by the reaction of .NO2 with the VA cation radical, rather than with the benzylic radical. The nitration of two other LiP substrates, 1,4-dimethoxybenzene (II) and tyrosine (III), also was examined. Product analysis of reactions conducted in the presence of H2O2 with these substrates indicated less nitrated product was formed from 1,4-dimethoxybenzene and no nitrated product was formed from tyrosine. However, significant amounts of nitrated products were formed from 1,4-dimethoxybenzene and tyrosine when glucose and glucose oxidase were used as an H2O2 source. These results suggest that a reductant, either the veratryl alcohol benzylic radical or superoxide, is required in the reaction to reduce TNM to generate .NO2. These results provide further evidence for the formation of the VA cation radical and the first chemical evidence for the formation of the VA benzylic radical in LiP-catalyzed reactions.

Relationship between NMR relaxation characteristics and water activity in cereal leaves.

The relationship of imposed water activity a(w) (equilibrium relative humidity) with conventional water status parameters and proton spin-lattice relaxation time T1 of leaf water was studied in pearl millet and wheat. The water activities of different levels were created by equilibrating the leaves in varying concentrations of PEG-6000 solutions. With decreasing a(w), relative water content and T1 decreased linearly and other variables (leaf water potential and leaf water content) decreased exponentially upto a dehydration level of a(w) approximately 0.978 for pearl millet, 0.986 for drought susceptible wheat var. HD2329 and 0.975 for tolerant wheat var. C306. Below that level there was abrupt reduction in all parameters except T1 which registered an increase. The changes in short and long components of T1 with changes in a(w) have also have been discussed for pearl millet.

Oxidation of dimethoxylated aromatic compounds by lignin peroxidase from Phanerochaete chrysosporium.

The stabilities of the cation radicals of veratryl alcohol, 3,4-dimethoxytoluene and 1,4-dimethoxybenzene were compared by monitoring the formation of dimeric products during the oxidation of these substrates by lignin peroxidase (LiP). LiP oxidized veratryl alcohol to generate veratraldehyde as the major product. Several other monomeric products were obtained in low yield. Dimeric products resulting from the coupling of two cation radicals, or a cation radical with a neutral molecule, were obtained only in trace amounts or not at all. This suggests that the cation radical of veratryl alcohol rapidly loses a benzylic proton to form a benzylic radical which undergoes further reactions to form veratraldehyde. In contrast, the LiP oxidation of 3,4-dimethoxytoluene generated the dimeric product 3-(2,3-dimethoxy-6-methylphenyl)-4-methyl-1,2-benzoquinone as the major product. Several other monomeric and dimeric products were produced in lower yields. The generation of these dimeric products indicates that the cation radical of 3,4-dimethoxytoluene is considerably more stable than that of veratryl alcohol. This suggests that the electronegative benzylic oxygen of veratryl alcohol increases the acidity of the benzylic protons, destabilizing the veratryl alcohol cation radical. LiP oxidized 1,4-dimethoxybenzene to generate 1,4-benzoquinone and 2-(2,5-dimethoxyphenyl)-1,4-benzoquinone as the major products. The formation of these products indicates that the cation radical of 1,4-dimethoxybenzene also is relatively stable, as previously demonstrated by ESR. All of these results indicate that the veratryl alcohol cation radical generated by LiP oxidation is unstable, suggesting that it would not act as a diffusable radical mediator in LiP-catalyzed reactions.

Aromatic nitroreductase from the basidiomycete Phanerochaete chrysosporium.

A membrane-associated aromatic nitroreductase activity was identified in cell-free extracts of the lignin-degrading fungus Phanerochaete chrysosporium. The enzyme catalyzed the nitro group reduction of 1,3-dinitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, 1-chloro-2,4-dinitrobenzene, and 2,4-dichloro-1-nitrobenzene. The corresponding hydroxylamines and/or amines were identified as reaction products by HPLC and/or GC-MS. 1-Nitroso-3-nitrobenzene and 1-hydroxylamino-3-nitrobenzene also were reduced by the enzyme, suggesting they were intermediates in the reaction. The enzyme required NAD(P)H as a cosubstrate and the optimal pH and temperature for the reaction were 6.5 and 50 degrees C, respectively. Enzyme activity was not observed in the presence of molecular oxygen. The membrane-associated enzyme could be solubilized with the nonionic detergent Triton X-100.

Oxidation of dibenzo-p-dioxin by lignin peroxidase from the basidiomycete Phanerochaete chrysosporium.

Dibenzo-p-dioxin (I) was rapidly degraded in ligninolytic cultures of the basidiomycete Phanerochaete chrysosporium. Lignin peroxidase (LiP) oxidized I to generate the following products: catechol (V), dibenzo-p-dioxin-2,3-quinone (VIII), 2-hydroxy-5-(2-hydroxyphenoxy)-1,4-benzoquinone (IX), 4,5-dihydroxy-1,2-benzoquinone (X), 2-(2-hydroxyphenoxy)-1,4-benzoquinone (XI), 4-hydroxy-1,2-benzoquinone (XII), and 1,2-benzoquinone (XIII). Identical products were formed when the reaction was conducted under argon. No incorporation of 18O into products was observed when the reaction was conducted under 18O2. Oxidation of I in H(2)18O resulted in incorporation of two atoms of 18O into the quinone VIII. Nonenzymatic hydrolysis of the quinone (VIII) yielded catechol (V), IX and X. Hydrolysis of VIII in H(2)18O resulted in incorporation of 18O atoms into IX and X, whereas no incorporation of 18O atoms into V was observed. These results are explained by mechanisms involving the one-electron oxidation of I by LiP to produce the corresponding cation radical. Nucleophilic attack of water on the cation radical generates a 2-hydroxydibenzo-p-dioxin radical, which is oxidized to a delocalized cation. The attack of water at position C-4a of the 2-hydroxydibenzo-p-dioxin cation, followed by oxidation and C-O-C bond cleavage, lead to formation of the quinone (XI), which undergoes 1,4-addition of water and cleavage of the second C-O-C bond to generate V and XII.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of a 1,2,4-trihydroxybenzene 1,2-dioxygenase from the basidiomycete Phanerochaete chrysosporium.

1,2,4-Trihydroxybenzene (THB) is an intermediate in the Phanerochaete chrysosporium degradation of vanillate and aromatic pollutants. A P. chrysosporium intracellular enzyme able to oxidatively cleave the aromatic ring of THB was purified by ammonium sulfate precipitation, hydrophobic and ion-exchange chromatographies, and native gel electrophoresis. The native protein has a molecular mass of 90 kDa and a subunit mass of 45 kDa. The enzyme catalyzes an intradiol cleavage of the substrate aromatic ring to produce maleylacetate. 18O2 incorporation studies demonstrate that molecular oxygen is a cosubstrate in the reaction. The enzyme exhibits high substrate specificity for THB; however, catechol cleavage occurs at approximately 20% of the optimal rate. THB dioxygenase catalyzes a key step in the degradation pathway of vanillate, an intermediate in lignin degradation. Maleylacetate, the product of THB cleavage, is reduced to beta-ketoadipate by an NADPH-requiring enzyme present in partially purified extracts.

Degradation of 2,4,5-trichlorophenol by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Under secondary metabolic conditions the white rot basidiomycete Phanerochaete chrysosporium rapidly mineralizes 2,4,5-trichlorophenol. The pathway for degradation of 2,4,5-trichlorophenol was elucidated by the characterization of fungal metabolites and oxidation products generated by purified lignin peroxidase (LiP) and manganese peroxidase (MnP). The multistep pathway involves cycles of peroxidase-catalyzed oxidative dechlorination reactions followed by quinone reduction reactions to yield the key intermediate 1,2,4,5-tetrahydroxybenzene, which is presumably ring cleaved. In the first step of the pathway, 2,4,5-trichlorophenol is oxidized to 2,5-dichloro-1,4-benzoquinone by either MnP or Lip. 2,5-Dichloro-1,4-benzoquinone is then reduced to 2,5-dichloro-1,4-hydroquinone. The 2,5-dichloro-1,4-hydroquinone is oxidized by MnP to generate 5-chloro-4-hydroxy-1,2-benzoquinone. The orthoquinone is in turn reduced to 5-chloro-1,2,4-trihydroxybenzene. Finally, the 5-chlorotrihydroxybenzene undergoes another cycle of oxidative dechlorination and reduction reactions to generate 1,2,4,5-tetrahydroxybenzene. The latter is presumably ring cleaved, with subsequent degradation to CO2. In this pathway, the substrate is oxidatively dechlorinated by LiP or MnP in a reaction which produces a quinone. The quinone intermediate is recycled by a reduction reaction to regenerate an intermediate which is again a substrate for peroxidase-catalyzed oxidative dechlorination. This pathway apparently results in the removal of all three chlorine atoms before ring cleavage occurs.

Degradation of 2,4-dinitrotoluene by the lignin-degrading fungus Phanerochaete chrysosporium.

Under ligninolytic conditions, the white rot basidiomycete Phanerochaete chrysosporium mineralizes 2,4-dinitrotoluene (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell extracts. The multistep pathway involves the initial reduction of I to yield 2-amino-4-nitrotoluene (II). II is oxidized by MnP to yield 4-nitro-1,2-benzoquinone (XII) and methanol. XII is then reduced to 4-nitro-1,2-hydroquinone (V), and the latter is methylated to 1,2-dimethoxy-4-nitrobenzene (X). 4-Nitro-1,2-hydroquinone (V) is also oxidized by MnP to yield nitrite and 2-hydroxybenzoquinone, which is reduced to form 1,2,4-trihydroxybenzene (VII). 1,2-Dimethoxy-4-nitrobenzene (X) is oxidized by LiP to yield nitrite, methanol, and 2-methoxy-1,4-benzoquinone (VI), which is reduced to form 2-methoxy-1,4-hydroquinone (IX). The latter is oxidized by LiP and MnP to 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (VII). The key intermediate 1,2,4-trihydroxybenzene is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial reduction of a nitroaromatic group generates the peroxidase substrate II. Oxidation of II releases methanol and generates 4-nitro-1,2-benzoquinone (XII), which is recycled by reduction and methylation reactions to regenerate intermediates which are in turn substrates for peroxidase-catalyzed oxidation leading to removal of the second nitro group. Thus, this unique pathway apparently results in the removal of both aromatic nitro groups before ring cleavage takes place.

Purification and characterization of E. histolytica antigen for diagnosis of amoebiasis.

Entamoeba histolytica soluble crude antigen was fractionated by gel filtration on Sephacryl S-300 into four fractions, viz. F1(669 kDa); F2(51.2 kDa); F3(25.1 kDa) and F4(10.5 kDa). F1 fraction was observed to be more sensitive and specific for the detection of antibody in amoebiasis than the crude and other fractions of purified antigens employing IHAT and ELISA. ELISA was found to be better than IHAT since it could detect antibody in the sera (3/6) of asymptomatic cyst passers. The cross reaction of crude antigen with toxocariasis (1/4) and toxoplasmosis (2/5) sera were associated with F4 fraction. F3 and F4 were having low molecular weight and were not sensitive in detection of antibody in amoebiasis. Biochemical characterization revealed glycoprotein nature of the specific (F1) antigen fraction.