Abstract
Activity of nitrate reductase (NR), the first enzyme in the nitrate-assimilation pathway, was estimated in the cotyledons of the sunflower( Helianthus annuus) using a standardized in-vivo method. Seedlings were grown in the light on a nitrate medium. Various factors that affect NR activity were optimized, including the molarity and pH of the reaction buffer, nitrate concentration, and use of a surfactant. We also determined whether NADH was required for nitrate reduction. The surfactant propanol (2%) gave the best results, and no NADH supplement was necessary: In a separate study, we compared the effect of various culturing components on in-vivo NR activity among monocot and dicot species, and found that Triton X-100 was the best surfactant for monocots whereas dicots performed better with n-propanol. Monocot species also required additional NADH as an external energy source. Moreover, specific purification procedures were needed to enhance NR activity in dicotyledons. Finally, we also assessed the efficacy of in-vivo versus in-vitro procedures for assaying monocots versus dicots.
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Literature cited
Aguera E, de la Haba P, Maldonado JM (1987) In vitro stabilization and tissue distribution of nitrogen assimilating enzymes in sunflower. J Plant Physiol128: 443–449
Aslam M, Buttery BR (1980) Anaerobic reduction of nitrate by plant leaves lacking in extractable nitrate reductase activity. Plant Physiol65: 57–62
Barber MJ, Notton BA (1991) Spinach nitrate reductase, effect of ionic strength and pH on the full and partial enzyme activities. Plant Physiol93: 537–540
Blondel AM, Blanc D (1975) Accidents vegetatifs sur argrummes en corse: Mise en euiadence dune carenca en molybdene par utilization du test de mesure in vivo de l’activite de la nitrate reductase. Ann Argon26: 277–287
Chanda SV, Joshi AK, Krishnan PN, Singh YD (1987) Invivo nitrate reductase activity in leaves of pearl millet,Pennisetum americana L. Leeke. Aust J Plant Physiol14: 125–134
Crawford NM, Campbell WH (1990) Fertile fields. Plant Cell2: 829–835
Doddema H, Telkamp GP (1979) Uptake of nitrate by mutants ofArabidopsis thaliana, disturbed in uptake or reduction of nitrate. ll, Kinetics. Physiol Plant45: 332–338
Echevarria C, Maurino SG, Maldonado JM (1984) Reversible inactivation of maize leaf nitrate reductase. Phytochemistry23: 2155–2158
Evans HJ, Nason A (1953) Pyridine nucleotide nitrate reductase from extracts of higher plants. Plant Physiol28: 233–254
Ferrari TE, Toder OC, Filner P (1973) Anaerobic nitrite production by plant cells and tissues: Evidence for nitrate pools. Plant Physiol51: 423–431
Fido RJ (1991) Isolation and partial amino acid sequence of domains of nitrate reductase from spinach. Phytochemistry30: 3519–3523
Hageman RH, Lambert RJ, Loussaert DH, Dalling MG, Klepper LS (1976) Nitrate and nitrite reductase as factors limiting protein synthesis,In Genetic Improvement of Seed Protein, National Academy Press, Washington, DC, 103–134
Hog K, Hartvigsen MB, Rasmussen OS (1983) Critical evaluation of thein vivo nitrate reductase for detection of two nitrate pools in wheat leaves. Physiol Plant59: 141–147
Jaworski EG (1971) Nitrate reductase assay in intact plant tissue. Biochem Biophys Res Commun43: 1274–1279
Johnson CB, Whittington WJ, Blackwood GC (1976) Nitrate reductase as a possible predictive test of crop yield. Nature262: 133–134
Lawrence JM, Herrick HE (1982) Media forin vivo nitrate reductase assay of plant tissue. Plant Sci Lett24: 17–26
Loomis WB (1974) Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Methods Enzymol31 A: 528–544
Mann AF, Hucklesby DP, Hewitt EJ (1979) Effect of aerobic and anaerobic conditions on thein vivo nitrate reductase assay in spinach leaves. Planta146: 83–89
Peuke AD, Tischner R (1991) Nitrate uptake and reduction of aseptically cultivated spruce seedlings,Picea abies (L.) Karst. J Exp Bot42: 723–728
Saroop S, Chanda SV, Singh YD (1999) Role of phytohormones on nitrate uptake onin vivo nitrate reductase activity ofBrassica juncea cotyledons. J Plant Nutr22: 1279–1290
Sood CR, Chanda SV, Singh YD (1996) Influence of plant growth regulators onin vivo andin vitro nitrate reductase activity of radish cotyledons. Acta Physiol Plant18: 287–294
Yamaya T, Oaks A, Boesel I (1980) Characterization of nitrate reductase inactivating protein obtained from corn roots and rice cell cultures. Plant Physiol65: 141–145
Yoneyama T (1981) Studies on thein vivo assay of nitrate reductase in leaves: Occurrence of underestimation of the activity due to dark assimilation of nitrate and nitrite. Plant Cell Physiol22: 1507–1520
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Chanda, S.V. Factors affecting nitrate reductase activity in some monocot and dicot species. J. Plant Biol. 46, 41–45 (2003). https://doi.org/10.1007/BF03030300
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DOI: https://doi.org/10.1007/BF03030300