Phospholipase D of rice bran. II. The effects of the enzyme inhibitors and activators on the germination and growth of root and seedling of rice

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Rice seeds contained fairly high level of phospholipase D activity. After seeding, the level of phospholipase D in seeds significantly increased both in specific activity and total activity in 15 randomly selected seeds. A dramatic increase in both activities was further observed when seeds were germinating and seedling and root were growing. Those non-germinating seeds and resting seeds under storage had only much lower level of phospholipase D activity. These data suggest that elevation in phospholipase D activities after seeding was one of the requirements for the seeds to germinate and for the growth of root and seedling. Polymyxin B sulfate, Protamine sulfate and EGTA strongly inhibited the activity of phospholipase D. In the presence of more than 0.34 mM of polymyxin B sulfate in the incubation buffer for germination both the germination and growth of root and seedling of rice were remarkably inhibited. In particular, the growth of root was almost entirely inhibited. While there was only slight inhibitory effect of EGTA and protamine sulfate on germination, the growth of root and seedling was also significantly inhibited. Thus, at a concentration of 0.1 mM, protamine sulfate also almost completely suppressed the growth of root. The specific activity value of phospholipase D found in root was only two thirds of that found in the absence of protamine sulfate in the incubation medium. Several divalent ions such as Ca 2+, Mg 2+, Sr 2+ and Ni 2+ were similarly tested for their effects on germination and growth of root and seedling. Ca 2+, Mg 2+ and Sr 2+ were activators of the enzyme whereas Ni 2+ was a strong inhibitor of the enzyme activity. These activators at a concentration of 5 mM in the incubation medium slightly stimulated the germination of rice seeds. By contrast, Ni 2+ almost entirely inhibited the germination and growth of root and seedling. While elevation in the activities of a number of enzymes during the germination is also possible, these results suggest that phospholipase D is at least one of the enzymes involved in germination process and growth of root and seedling of rice and that there is a correlation between the level of phospholipase D activity, and germination and growth of root and seedling of rice seeds.