NADP-malic enzyme: immunolocalization in different tissues34 plant maize and the C3 plant wheat

Abstract

In situ immunolocalization and Western blot analysis of separated cellular and subcellular fractions, were used to determine the localization of different isoforms of NADP-malic enzyme in both wheat (C 3 ) and maize (C 4 ) plants. In both techniques, an affinity purified anti(maize 62 kDa NADP-ME) IgG from the maize green leaf isoform also reacted with a 72 kDa protein in tissues of C 4 plants as well as C 3 plants. The light-inducible 62 kDa isoform is located in bundle sheath chloroplasts of maize leaves. In etiolated leaves and in roots of maize there is evidence for the occurrence of a 72 kDa isoform which co-migrates on 2-D (SDS and isoelectric focusing) PAGE. The 72 kDa isoform is also present in low levels in green leaves. This form may occur in multiple intracellular compartments; but in situ immunolocalization experiments and Western blot and activity assays on fractionated protoplasts indicate that a significant amount of this isoform occurs in plastids. With regards to C 3 plants such as wheat, a 72 kDa isoform in leaves is largely confined to the chloroplasts based on in situ immunolocalization and Western blots and enzyme activity assays with fractionated protoplasts. In maize, it appears that the constitutive expression pattern of a possible C 3 ancestral gene for NADP-malic enzyme has been maintained, and a high level expression of a light-inducible isoform located in bundle sheath chloroplasts (62 kDa) has been acquired during its evolution.