Iron-Stress Induced Redox Activity in Tomato

Tomato plants (Lycopersicum esculentum Mill.) were grown for 21-days in a complete hydroponic nutrient solution including Fe3+ethylenediamine-di(o-hydroxyphenylacetate) and subsequently switched to nutrient solution withholding Fe for 8 days to induce Fe stress. The roots of Fe-stressed plants reduced chelated Fe at rates sevenfold higher than roots of plants grown under Fesufficient conditions. The response in intact Fe-deficient roots was localized to root hairs, which developed on secondary roots during the period of Fe stress. Plasma membranes (PM) isolated by aqueous two-phase partitioning from tomato roots grown under Fe stress exhibited a 94% increase in rates of NADH-dependent Fe3 -citrate reduction compared to PM isolated from roots of Fe-sufficient plants. Optimal detection of the reductase activity required the presence of detergent indicating structural latency. In contrast, NADPH-dependent Fe3+-citrate reduction was not significantly different in root PM isolated from Fe-deficient versus Fe-sufficient plants and proceeded at substantially lower rates than NADH-dependent reduction. Mg2 -ATPase activity was increased 22% in PM from roots of Fe-deficient plants compared to PM isolated from roots of Fe-sufficient plants. The results localized the increase in Fe reductase activity in roots grown under Fe stress to the PM. Iron deficiency induces a series of adaptive reactions in roots of Fe-efficient plants (10, 26). Roots under Fe stress show a dramatic increase in root hairs and rhizodermal transfer cells (16, 17), have a 5to 10-fold increased capacity to reduce chelated Fe (4, 1 1) and show increased acidification of the rhizosphere (13, 27). Both acidification and Fe reductase activities are spatially coincidental on the newly developed roots (27). In an early attempt to explain reduction of Fe by roots, Brown and Ambler (6) and Olsen et al. (22, 23) proposed the secretion of reductants into the rhizosphere. In support of this hypothesis, they as well as Romheld and Marschner (25) detected Fe reducing compounds, identified as o-diphenolics, in the rhizosphere of Fe-stressed roots. However, these compounds can reduce high-affinity Fe3+-chelates only very slowly (1 1, 25). 'Supported in part by U.S. Department of Agriculture grant 8837231-3916. 2 Present address: Foreign Disease-Weed Science Research, USDAAgricultural Research Service, Building 1301, Fort Detrick, Frederick, MD 21701. An alternate mechanism of Fe-stress induced Fe reduction has been presented by Chaney et al. (11) and subsequently expanded upon by Bienfait et al. (3, 4). In their hypothesis, a transplasma membrane (PM3) electron transport chain supplies electrons from cytoplasmic reducing equivalents to the extracytoplasmic cell surface where Fe reduction occurs. Since the proposal of an Fe-stress-responsive electron transport chain by Chaney et al. (1 1), evidence for PM redox systems and their involvement in Fe reduction has become substantial. For example, a number of reports suggest the presence of NAD(P)H dehydrogenase activity on plant PM (reviewed in ref. 12). The presence of a PM-associated redox system in plants has been demonstrated in intact roots, whole cells, protoplasts, isolated membranes, and purified PM preparations (reviewed in ref. 12). Also, the reduction of Fe at the cell surface is enzymatic (1, 4, 25), occurs on the extracytoplasmic surface of the PM (2) and is increased circa 10-fold during Fe stress (4, 1 1). The structure of the PM Fe3+-chelate reductase is largely unknown. Sijmons et al. (29) presented evidence indicating the involvement of cytoplasmic reduced pyridine nucleotides, possibly NADPH, as the source of reducing equivalents. Sijmons et al. (28) demonstrated that the addition of ferricyanide or Fe3+-EDTA to intact roots caused a rapid, 30 to 40 mV depolarization of the membrane potential, suggesting a transmembrane flow of electrons. Cakmak et al. (9) reported the nonobligatory involvement of superoxide radicals in Fe reduction and, based on theoretical consideration, proposed a semiquinone in the 1-electron reduction of 02. Such a mechanism differs significantly from the superoxide generating system on the PM of leucocytes, which involves a flavinmediated transfer of electrons to a b-type Cyt (20). Until recently, investigations into the mechanism of Fe reduction at the PM and the mechanism of reductase induction during Fe stress adaptation have centered around intact roots. We report here evidence for the localization of the Festress redox response in a tomato root PM fraction prepared by aqueous two-phase partitioning. 'Abbreviations: PM, plasma membrane(s); BPDS, bathophenanthrolinedisulfonate; EDDHA, ethylenediamine-di(o-hydroxyphenylacetate); FCR, ferricyanide reductase; HEDTA, N-hydroxyethylethylenediaminetriacetic acid; PDTS, 3-(2-pyridyl)-5,6-diphenyl1,2,4triazine sulfonate.