Activity and localization of lipoxygenase in the ovule of Larix kaempferi (Lamb.) Carr. during megasporogenesis

Abstract

The anatomical structure of the ovule of Larix kaempferi is typical for gymnosperms. During megasporogenesis, three megaspores arranged in one line are formed after incomplete meiotic division. Lipoxygenase occurrence in the Japanese larch ovules was shown by the Western-blotting method and confirmed using native PAGE. The localization of LOX in the young ovule of L. kaempferi during megasporogenesis was analysed by the immunogold labelling technique. The activity of the enzyme was measured spectrophotometrically at 234 nm using linoleic acid as a substrate. The location of LOX was rather random; however, clusters of immunogold particles were often observed in the vacuoles of integument cells. It was found in the cytoplasm, cell walls, vacuoles, starch grains, and nuclei. LOX activity in developing ovules of the Japanese larch was not correlated with the number of antibody-labelled molecules of the enzyme, which may indicate its non-enzymatic function. Two optima for LOX activity, at pH 8.0 and pH 7.0, were found in L. kaempferi developing ovules. The enzyme with a maximum activity at pH 8.0 appeared in ovules with a megaspore mother cell as well as with a triad of megaspores; the second maximum activity (at pH 7.0) was detected only at the stage with the triad of megaspores in the nucellus.