Gibberelic acid-mediated activation of monophenolase in de-embryonated half-seeds of wheat ( Triticum Aestivum)

Abstract

Stimulation of monophenolase activity was observed when de-embryonated prewashed half-seeds of wheat were imbibed in a solution of gibberellic acid (GA 3, 10 t̄5 M). Crude extracts, prepared from GA 3-treated half-seeds, showed ca a two-fold increase in monophenolase activity over the controls at pH 6.6, while a dramatic rise in enzyme activity (seven- to nine-fold) was observed at pH 9.0. The partially purified (NH 4) 2SO 4 fraction precipitate (30–50% saturation) also showed enhancement of enzyme activity at pH 9.0 in GA 3-treated half-seeds, while in controls, there was negligible activity at this pH. Administration of five amino acid analogues (1 mM each) to half-seeds showed no significant inhibition of GA 3-stimulated monophenolase activity, but proved very effective in decreasing (86% inhibition) the GA 3-induced amylase activity. This indicated that the hormone-regulated monophenolase activity was not dependent on de novo protein synthesis. Treatment of half-seeds with GA 3 modified the monophenolase and altered the electrophoretic pattern. The enzyme was relatively thermostable at 55° and the pH optimum was shifted from pH 7.0 to 9.0. In addition, the GA 3-treated half-seeds showed relatively high stability of enzyme activity in the presence of (NH 4) 2S0 4 ions. These alterations in the GA 3-stimulated nomophenolase suggest there is activation of preformed enzyme molecules. The appearance of slow-migrating multiple forms on acrylamide gels, in response to GA 3 treatment, is probably due to the association of fast-migrating forms. Such oligomerization could result in a conformational change leading to enzyme activation. This may be an adaptive mechanism so that the enzyme can function with varying temperature, pH and ionic strength during early stages of seed germination.