REGULATION OF NITRATE REDUCTASE ACTIVITY IN SPINACH {Spinacia oleracea L.) DURING LIGHT AND DARK TRANSITION

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Date
2005
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MPKV, UNIVERSITY LIBRARY RAHURI
Abstract
The enzyme nitrate reductase (NR, EC 1.6.6.1) was determined from the leaves of spinach seedlings by in vivo and in vitro methods. Various experiments were performed with the leaves of 10-day-old seedlings of spinach. The spinach plants were grown in the pots filled with medium black calcareous soil under natural daylight condition during October, 2004 to February, 2005. The standardized infiltration medium (5.0 ml) for maximum in vivo NR activity in plus KNO3 assays in spinach leaves was : Sodium phosphate buffer, 100 mM, pH 7.5; KNO3, 100 mM and n-propanol 4% (v/v). The enzyme was extracted from the leaves in an extraction buffer containing 100 mM Hepes buffer (pH 7.5), 5 mM DTT, 1 mM EDTA and (0.1%, v/v) Triton-X-100. The reaction mixture in a total volume of 2.2 ml contained in mM : Hepes buffer (pH 7.5), 50; KNO3, 10; NADH, 0.1 and crude enzyme extract, 0.2 ml. The in vitro reaction rate was linear with time over a 30-min incubation period. In spinach leaves, in vivo nitrate reductase activity declined gradually under dark and significant decrease in the activity was noticed only after 60 min of darkness. When one hr dark treated plants were exposed to natural daylight conditions, in vivo NR activity gradually increased. In vivo NRA declined slowly upto 60 min of darkness in presence of Mg2+, however, the decline was less pronounced in the absence of Mg2+. When the potted plants were transferred from natural light to dark, the nitrate content in spinach leaves increased gradually upto 60 min of darkness. However, when these potted plants were again shifted to natural light, a gradual decline in nitrate content was noticed. When one hr dark treated plants were transferred to light, a gradual decline in nitrate content was observed upto 60 min. In case of in vitro NR activity in leaves of potted plants kept in light for one hr and subsequently shifted to darkness, a gradual decline was observed upto 1 hr. When these plants were again shifted to natural light, a gradual increase in the activity was
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