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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