Early response to gibberellic acid of monophenolase (tyrosinase) activity in de-embryonated half-seeds of wheat

Abstract

A significant enhancement (7–8 fold) of monophenolase activity by GA 3 was observed in de-embryonated half-seeds of wheat after 2 hr of imbibition. This early response to GA 3 (2–12 hr) was apparent only after lowering the endogenous levels of phytohormone in wheat half-seeds by leaching. Cycloheximide (CHI, 20 μg/ml) failed to inhibit GA 3-stimulated (GA 3, 10 μM) monophenolase activity. This ruled out the requirement of de novo protein synthesis for the hormone-regulated activity of monophenolase. Abscisic acid (100 μM) effectively stopped the GA 3-mediated activation of monophenolase. Curiously, the early stimulatory response to GA 3 of monophenolase activity was completely mimicked by the administration of phosphate ions (Pi, 75 mM). Simultaneous addition of GA 3 and Pi to wheat half-seeds, however, showed no cumulative effect on the activation of monophenolase. Both GA 3 and Pi activated monophenolase exhibited altered molecular properties, in terms of shift in the pH optimum of enzyme activity towards alkalinity (pH 9.0) and relatively increased thermostability of the enzyme at 50°. Molecular sieving of the enzyme fraction on Sephacryl S-200 revealed a single molecular form of monophenolase ( M r 45 000) both in control and GA 3-treated wheat half-seeds during the first 12 hr of imbibition. This indicated that the activation of monophenolase by GA 3 is due to some structural modification of the enzyme without its oligomerization. We envisage that the modulation of monophenolase activity by GA 3 during early imbibition of wheat half-seeds is a true primary response to the phytohormone.