Purification and properties of lipoxygenase induced in downy mildew resistant pearl millet seedlings due to infection with Sclerospora graminicola

Abstract

Induction of lipoxygenase (LOX) was studied in pearl millet seedlings upon inoculation with Sclerospora graminicola. Resistant pearl millet seedlings exhibited a 2.4-fold increase in LOX activity after inoculation with the downy mildew pathogen S. graminicola. This increase was mainly due to the synthesis of a new LOX isozyme designated as LOX-6. Three of the six isozymes of lipoxygenase designated LOX-1, -3 and -6, were purified from inoculated downy mildew resistant pearl millet seedlings as electrophoretically homogeneous proteins. The isozymes were purified stepwise by ammonium sulfate fractionation, DEAE-sephadex A-50 and sephadex G-200 gel-filtration. Purification factor for LOX-1, -3 and -6 were 46.5, 73.6 and 115.7, respectively. The purified LOX-1, -3 and -6 had molecular weight of 83, 77 and 73 kDa with a pI of 5.5, 5.8 and 6.2, respectively. The results indicate that the LOX isozymes were dimers composed of two unequal subunits of 43 and 40 for LOX-1, 40 and 37 for LOX-3, 38 and 35 for LOX-6. The optimum pH for LOX 6 was 6.5 being stable from pH 4.5 to 8.0. The LOX-1 and -3 had a similar optimum pH of 9.0. LOX isozymes had a different thermal stability ranging from 0 to 30°C. Esculetin, NDGA, SHAM, N-propylgallate and copper sulfate were found to be the potent inhibitors of all three isozymes of LOX. All the isozymes of LOX revealed similar reaction with the metal ions like Ca²⁺, Ba²⁺, Mn²⁺, Cu²⁺, Fe²⁺ and Mg²⁺ with very few exceptions. Butyl hydroxyanisole caused the strongest inhibition of the pearl millet LOX isozymes among the various antioxidants tested. The LOX isozymes showed preferential activity towards linoleic acid followed by linolenic acid as substrate.