Activation of plasmalemmal NADPH oxidase in etiolated maize seedlings exposed to chilling temperatures |
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Authors: | M S Piotrovskii T A Shevyreva I M Zhestkova M S Trofimova |
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Institution: | 1.Timiryazev Institute of Plant Physiology,Russian Academy of Sciences,Moscow,Russia |
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Abstract: | Five-day-old etiolated seedlings of maize (Zea mays L.) were used to study the kinetics of hydrogen peroxide formation upon lowering growth temperature from 25 to 6°C. The total
content of hydrogen peroxide in root and shoot tissues increased by 30–40% after 2-h cooling compared to the control level
but returned to the initial level or decreased even lower after 24-h cooling. In order to prove the involvement of plasma
membrane NADPH oxidase in changes of hydrogen peroxide content upon cooling, isolated plasma membranes were obtained from
untreated plants and from seedlings chilled at 6°C for 2 and 24 h. The NADPH-dependent generation of superoxide anion radical
in isolated plasma membranes was quantified by measuring the rate of formazan production from the tetrazolium salt XTT. The
activity of plasma membrane NADPH oxidase in shoots was 50 ± 9 nmol O2/(mg protein min), which was 1.5 times higher than the activity in roots. The enzyme activity in plasma membranes was inhibited
by low concentrations of diphenyleneiodonium. The effective concentration EC50 was 5.10 μM for shoots and 9.05 μM for roots. The activity of plasma membrane NADPH oxidase increased after 2-h cooling of
seedlings but reversed to the control level after 24-h cooling. This transient activation of NADPH oxidase upon cooling was
similar to the pattern of hydrogen peroxide formation in shoots and roots. Analysis of NADPH oxidase activity of plasma membrane
proteins after their separation in denaturing conditions followed by subsequent renaturation revealed four diphenyleneiodonium-sensitive
bands with mol wt of 130, 88, 51, and 48 kD. Western blot analysis of the reaction with antibodies against the catalytic domain
of phagocyte NADPH oxidase revealed the proteins with mol wt of only 88 and 48 kD. The properties of molecular organization
of plasma membrane NADPH oxidase are discussed in terms of its role in cell signaling. |
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