Fe³⁺-Chelate Reductase Activity of Plasma Membranes Isolated from Tomato (<i>Lycopersicon esculentum</i> Mill.) Roots

Abstract

Reduction of Fe(3+) to Fe(2+) is a prerequisite for Fe uptake by tomato roots. Ferric chelate reductase activity in plasma membranes (PM) isolated from roots of both iron-sufficient (+Fe) and iron-deficient (−Fe) tomatoes (Lycopersicon esculentum Mill.) was measured as NADH-dependent ferric citrate reductase and exhibited simple Michaelis-Menten kinetics for the substrates, NADH and Fe(3+)(citrate(3−))(2). NADH and Fe(3+)(citrate(3−))(2) K(m) values for reductase in PM from +Fe and −Fe tomato roots were similar, whereas V(max) values were two- to threefold higher for reductase from −Fe tomatoes. The pH optimum for Fe-chelate reductase was 6.5. Fe-chelate reductases from −Fe and +Fe tomato roots were equally sensitive to several triazine dyes. Reductase was solubilized with n-octyl β-d-glucopyranoside and electrophoresed in nondenaturing isoelectric focusing gels. Three bands, with isoelectric points of 5.5 to 6.2, were resolved by enzyme activity staining of electrofocused PM proteins isolated from +Fe and −Fe tomato roots. Activity staining was particularly enhanced in the isoelectric point 5.5 and 6.2 bands solubilized from −Fe PM. We conclude that PM from roots of +Fe and −Fe plants contain Fe-chelate reductases with similar characteristics. The response to iron deficiency stress likely involves increased expression of constitutive Fe-chelate reductase isoforms in expanding epidermal root PM.