ASSESSMENT OF NITRATE REDUCTASE POTENTIAL AND CARBON MONOXIDE SENSITIVITY OF CYTOCHROME c OXIDASE IN THE LEAVES OF CYTOPLASMIC MALE STERILE AND MAINTAINER SEEDLINGS OF PEARL MILLET, Pennisetum glaucum L.
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Date
2000
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Mahatma Phule Krishi Vidyapeeth, Rahuri.
Abstract
Seedlings of CMS and maintainer lines of pearl millet
were grown in soil-filled pots in natural daylight under the green
house conditions in the Department of Biochemistry, Mahatma Phule
Krishi Vidyapeeth, Rahuri during the months of March to August
1999 to assess the actual and potential nitrate reductase (NR) activity
in the leaves. The laboratory experiments were carried out for the
standardization of NR enzyme from the pearl millet system. The
differences in the redox state of cytochrome a3 during in situ respiration of leaves of CMS and maintainer lines of pearl millet were
also examined by an in vivo CO-aerobic assay of NR by sparging CO
for one min through leaf segments and by exposing the leaf segments
to a defined CO:02 ratio of 5.
The in vivo NR assays for leaf materials of 10-day-old
pearl millet seedlings were optrndzed in terms of substrate and
n-propanol concentrations added into the infiltration media. The
optimum concentration of KNO3 added into the infiltration media,
required for maximum in vivo NRA, was found to be 100 mM in pearl
millet system for the determination of in vivo NRA.
When the leaf materials of 10-day-old CMS and
maintainer lines were examined for in vivo NR potential, the enzyme
activity was higher in the assays where exogenous nitrate (100 mM)
was added (plus KNO3 assays) than that of assays performed without
nitrate (minus KNO3 assays). The leaf materials of three CMS and one
maintainer lines of pearl millet were examined for carbon monoxide
sensitivity of cytochrome c oxidase by sparging CO for one min
through weighed leaf fragments (2.5 cm size) and then incubating
under aerobic condition for determination of NO2 accumulated in
vivo. All the lines were found to be sensitive to CO by this
technique. Further, a precisely defined CO:02 ratio of 5 appropriate for assessing the redox state of cytochrome as in the
leaves of CMS and maintainer lines of pearl millet.
These studies thus revealed that rather than sparging CO
for one min through leaf segments and incubation under aerobic
conditions, the technique of exposing leaf segments to a precisely
defined CO:C>2 ratio of 5 appears more appropriate in assessing the
redox state of cytochrome a3 as reflected in the significant amount of
nitrite accumulated in the leaves of pearl millet lines.
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