The nitrate reductase

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It is reported that nitrate reductase is an important enzyme of nitrogen metabolism. It is inducible by No3 contents Huffaker,et al(1982). Under drought conditions, only inoculation with exotic carbuncular mycorrhizal fungi increased shoot and root nitrate reductase activity 188% and 38% respectively with respect to plants neither inoculated nor treated with compost. Alguacil, et al. (2006). It is also concluded that nitrate supply to the leaves from roots play a much larger regulatory role in controlling nitrate reductase activity in plants. Nitrate reductase activity in maize leave was consistent with the well known fact that nitrate increases and NH4 decreases nitrate reductase active( Dale, et al.1976., David, et al. 1983)

Pace et-al(1990)The results demonstrate that reduction of entering nitrate by roots as well as shoots was regulated by concurrent photosynthesis. Although in vitro nitrate reductase activity of both tissues declined by 60% during a 10-hour period of CO2 stress, the remaining activity was greatly in excess of that required to catalyze the measured rate of 15NO3- reduction. Root respiration and soluble carbohydrate levels in root tissue were also decreased by CO2 stress. Collectively, the results indicate that nitrate uptake and reduction were regulated by the supply of energy and carbon skeletons required to support these processes, rather than by the potential enzymatic capacity to catalyze nitrate reduction, as measured by in vitro nitrate reductase activity. (Konard, et al., 1983). Widman, et al. (1993) also concluded that nitrate reductase activity of plants species was reduced in nitrate deficient soil.

Soraya, et al. (2007) found that cells halotolerant cyanobactarium grown in nitrate containing medium showed higher nitrate reductase activity than the cells grown in glutamine medium. Nitrate reductase levels are also regulated by denovo synthesis and protein degradation (David, et al., 1983). Andrew, et al. (1980) reported that nitrate reductase activity was higher in comparable leaves from the high than from the low nitrate reductase genotype through the grain development period. There was no correlation between nitrate reductase activity and nitrate content of leaves. However, high nitrate reductase genotype maintained higher amount of nitrate during later stage of grain development.

The NR activities were maximum in 100% NPK followed by organically treated plots. This was attributed to supply of readily available nitrate from inorganic fertilizers to the plants while in organically treated plots, nitrate release was comparatively slow. Further, in the organically treated plots ammonium ions are released after decomposition of organic matter and the ammonium ions so released are inhibitory to the NR activity (Claussen and Lenz, 1999). Thus the results clearly showed that all the organic manures used in the study played a significant role in increasing root and shoot biomass, chlorophyll content of rainy season crops (i.e., soybean and sorghum) and the subsequent wheat crop in the winter season for all the three cropping systems.

Alguacil (2006) reported that compost addition had no significant effect on NR activity in shoots of J. oxycedrus seedlings, under either well-watered or drought-stressed conditions. The seedlings inoculated with exotic AM fungi showed the highest values of shoot NR activity in drought stress conditions. Compost addition and exotic AM fungi inoculation increased significantly the NR activity in roots. The water stress decreased NR activity in roots of J. oxycedrus plants grown in the soil amended with compost.

Foyer et al(1998)A correlation between maximal extractable foliar nitrate reductase (NR) activity and the rate of CO2 assimilation was observed. The NR activation state and maximal extractable NR activity declined rapidly in response to drought. Photosynthesis and NR activity recovered rapidly when nutrient solution was restored at this point. The decrease in maximal extractable NR activity was accompanied by a decrease in NR transcripts, whereas Suc phosphate synthase and phosphoenolpyruvate carboxylase mRNAs were much less affected. The coordination of N and C metabolism is retained during drought conditions via modulation of the activities of Suc phosphate synthase and NR commensurate with the prevailing rate of photosynthesis.

Konard et-al(1983)observed that Nitrate reductase activity in leaves was consistent with the well known fact that N03- increases, and NH4+ and amide-N decrease, nitrate reductase activity. In roots, nitrate reductase activity in vitro was correlated with the rate of nitrate reduction in vivo. Inasmuch as nitrate reduction results in the production of OH- and stimulates the synthesis of organic anions,it was postulated that nitrate reductase activity of roots is stimulated by the released OH- or by the synthesized organic anions rather than by nitrate itself. Addtion of HCO3- to nutrient solution of maize seedlings resulted in a significant increase of the nitrate reductase activity in the roots. As HCO3-, like OH-, increases pH and promotes the synthesis of organic anions, this provides circumstantial evidence that alkamne conditions and/or organic anions have a more direct impact on nitrate reductase activity than do N03-, NH4-N, and amide-N.

It is also reported that NRA in plant tissue is proportional to nitrate concentration in soil solution and its synthesis is regulated by flux rather than leaf nitrate contents.(Naqvi et al.,1996).Shanker et al;(2001) reported that there is positive correlation between NRA and total organic nitrogen.Wang et al; (2004) concluded that Nitrate regulates nitrate reductase transcription,translocation and activation in higher plants.Hageman and Flesher, (1960) reported that NR is induced by No2.Ben Zioni et al.,(1971)proposed mechanism how No3 reduction in the shoots may increase the absorption of No3 by the roots.They suggested that after KNO3 translocation to shoots from the roots ,a stoichometric amount of malate is produced for the No3 reduction in the shoots.After the malate is synthesized , part of it moves downto the roots system as K -malate, where it is oxidized ,yielding KHCo3, which exchanges for KNo3 in the external medium. Thus No3 reduction in shoots promotes preferential uptake of No3 by the roots.

NPK Concentration

Prasad and Sinha (2000) investigated that the K, Zn, Cu, Fe and Mn requirement of rice and wheat could be met by the incorporation of crop residues. Recycling of crop residues can replenish 37% of the N removal by rice and wheat, 18% of P removal by wheat and 28% of P removal by rice in calcareous soil. Available N, K, S, Zn, Fe, Cu, Mn, B and Mo increased when different levels of fertilizers were applied along with FYM and crop residues. Gondhk and Mazzar (2005) reported that mineral and organo-mineral fertilizer resulted in decline of organic content in soil whereas FYM increased organic C in soil. Application of organic-mineral fertilizer increased available Zn content. Nardi et al. (2004) investigated evaluated that forty years of FYM manure application sustained total organic carbon (TOC) in the top layers while mineral treatments alone or mixed with FYM showed a minor effect on the organic matter evolution in a maize system.

NPK content in maize leaves and grain were also increased significantly with the application of organic and inorganic fertilizers (Sial, et al., 2007).

Crop Residues and Soil Properties

Ranjan, et al. (2007) reported that combined use of NPK and FYM increased SOC, total N, Olsen P and ammonium acetate extractable K by 47 %, 31% and 73% receptively. It also showed the highest levels of soil microbial biomass C and dehydrogenase activity.The use of organic sources along-with inorganic fertilizers also improves soil physical conditions. Prasad and Sinha (2000) found that soil aggregation and soil porosity-hydraulic conductivity was increased with integrated nutrient management. Total porosity of soil and water holding capacity were increased with the application of both FYM and inorganic fertilizer. In 0-15 cm soil layer, the total porosity significantly increased with FYM over control (Rehana et al. 2007). Application of 5 t FYM ha-1 every year significantly improved the soil organic content (Petal et al., 2008).


Enzyme Extraction

For extracting antioxidant enzymes, fresh leaves (0.5 g) were ground using a tissue grinder in 5 ml of 50 mM cooled phosphate buffer (pH 7.8) placed in an ice bath. The homogenate was centrifuged at 15000 x g for 20 min at 4 °C. The supernatant was used for determining the activities of enzymes.

Superoxide dismutase: (SOD)

The activity of SOD was determined by measuring its ability to inhibit the photoreduction of nitroblue tetrazolium (NBT) following the method of Giannopolitis and Ries (1977). The reaction solution (3 ml) contained 50 µM NBT, 1.3 µM riboflavin, 13 mM methionine, 75 nM EDTA, 50 mM phosphate buffer (pH 7.8), and 20 to 50 µl of enzyme extract. The test tubes containing the reaction solution were irradiated under light (15 fluorescent lamps) at 78 µmol m-2 s-1 for 15 min. The absorbance of the irradiated solution at 560 nm was read using a spectrophotometer (IRMECO, U2020). One unit of SOD activity was defined as the amount of enzyme that inhibited 50% of NBT photoreduction.

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