Iron stress-induced redox reactions in bean roots

Iron stress-induced and constitutive redox activity of bean ( Phaseolus vulgaris L. cv. Delinel) roots was measured on intact plants using FeEDTA and ferricyanide as electron acceptors. The presence of the translation inhibitor cycloheximide caused a decrease in the reduction of both oxidants. However, a differential decline in the reduction rates of FeEDTA and ferricyanide was observed, suggesting enzyme heterogeneity. In the presence of the H⁺ -ATPase inhibitor vanadate, the reduction of FeEDTA was nearly completely suppressed in both Fe-deficient (–Fe) and Fe-sufficient (+Fe) plants, providing evidence for an involvement of plasma membrane-bound ATPase activity in the regulation of the reduction process. The inhibition of the ferricyanide reduction by vanadate was restricted to –Fe plants.
The data are interpreted in terms of simultaneous operation of distinct redox systems in roots of iron-deficient bean plants. The role of proton extrusion in iron stress-induced electron transfer is discussed.     

Root-mediated ferric reduction-responses to iron deficiency, exogenously induced changes in hormonal balance and inhibition of protein synthesis

The effects of the auxin analogue 2,4-dichloro-phenoxyaceticacid (2,4-D), the translation inhibitor cycloheximide (CHM)and iron deficiency on electron transport activities in rootshave been investigated. Deprivation of an external Fe supplycaused an increase in capacity to reduce both FeEDTA and theartificial electron acceptor ferricyanide. With respect to theiron stress-induced redox activity, bean (Phaseolus vulgarisL.) roots reduced both substrates at equal rates, while rootsof Plantago lanceolata L. exhibited clearly higher rates withFeEDTA. The iron stress induced FeEDTA and ferricyanide reductionactivities were unequally affected by CHM in both species, reflectingthe differences in substrate specificity. Application of 2,4-D via the nutrient solution induced an increasein ferricyanide reduction rates in iron-sufficient roots ofP. lanceolata; the reduction of FeEDTA was only slightly affected.The presence of 2,4-D caused no further enhancement in reductionactivity of plants grown without Fe. However, iron-deficientplants exhibited a distinct developmental pattern of ferricreduction activities when treated with 2,4-D, resembling thedevelopment of 2,4-D-induced ferricyanide reduction by iron-sufficientplants. This information is taken as support that the reduction of ferricyanideand FeEDTA can in part be separately regulated. The data alsoseem to indicate that the amount of Fe²⁺ is the main cause forinduction or repression of ferric chelate reduction by rootsof iron-efficient plants. Key words: Phaseolus vulgaris, Plantago, ferric iron reduction, cycloheximide, 2,4-dichlorophenoxyacetic acid     

Effects of various inhibitors on in vivo reduction by Plantago lanceolata L. roots

Roots of Plantago lanceolata L. showed an iron stress-induced increase in the rates of electron transport to the extracytoplasmatic acceptors FeEDTA and ferricyanide. No significant changes in the reduction of hexachloroiridate were observed with respect to the iron-nutritional status of the plants. The reduction activity of iron-deficient roots was inhibited by the translation inhibitor cycloheximide (CHM) and the amino acid analog p-fluorophenylalanine (FPA). In both cases, the reduction of FeEDTA and ferricyanide was affected to a different extent, providing evidence for enzyme heterogeneity. Resupply of FeEDTA to iron-deficient plants resulted in a qualitatively similar pattern of decrease in FeEDTA and ferricyanide reduction rates, although a longer time period was required for the decrease of the redox activity by iron resupply compared to the effect of inhibitors of protein synthesis.Inhibitors of the plasma membrane (PM)-bound H⁺-ATPase decreased the FeEDTA reduction activity of iron-deficient plants. In contrast, the reduction of ferricyanide and hexachloroiridate was not inhibited. Oxidation of ferrocyanide occurs in both iron-deficient and iron-sufficient plants at comparable rates. The reaction was decreased by the H⁺-ATPase inhibitor orthovanadate.The results are interpreted in terms of a simultaneous action of distinct redox systems in iron-deficient roots. The role of proton extrusion in the regulation of iron stress-induced electron transport is discussed.     

Pyridine nucleotide pool size changes in iron-deficient Plantago lanceolata roots during reduction of external oxidants

The involvement of pyridine nucleotides in the reduction of extracytoplasmatic electron acceptors by iron-deficient Plantago lanceolata L. roots has been examined by measuring the changes in NAD(P)H and NAD(P)⁻ induced by various external acceptors. Exposure of the plants to FeEDTA, ferricyanide, ferric citrate or hexachloroiri-date resulted in a transient decrease in NADPH and an increase in NAD⁻. No major differences in this pattern were observed between acceptors which were assumed to be reduced by different enzymes. The application of the membrane-permeable oxidant nitro blue tetrazolium led to similar changes in reduced and oxidized pyridine nucleotides and decreased the reduction of external acceptors. The amino acid analog p -fluorophenylalanine caused a transient decline in both NADPH level and NADPH/ NADP⁻ ratio and a decrease in the ratio of NADH to NAD⁻ without affecting the level of NADH. Exposure of the plants to the translation inhibitor cycloheximide increased both NADH and NADPH concentrations. A comparison of the redox activities and pyridine nucleotide fractions after inhibitor treatment revealed that the constitutive, but not iron stress-induced redox activity correlates with NADPH levels. These results are interpreted as confirming that the redox systems on the root plasma membrane are separately regulated. Possible metabolic reactions during the reduction processes are discussed.     

Reduction of root iron in Plantago lanceolata during recovery from Fe deficiency

Changes in root-associated Fe(III) reductase activity and Fe concentration during recovery from temporary iron starvation were investigated in hydroponically grown Plantago lanceolata L. Within two days, interruption of the Fe supply resulted in enhanced rates of reduction by intact plant roots. Transfer of iron deficient plants to a solution containing various amounts of chelated Fe caused a transient increase in reduction activity before the rates declined to a level determined by the amount of Fe added. Repression of root-associated redox activity was independent of the Fe concentration in the preculture. When iron deficient plants were submitted to a supply of Fe localized to a part of the root system (split-root plants), the decrease in reduction rates was much more pronounced in the Fe-deprived portion of the roots than in the Fe-supplied one. No correlation was observed between root Fe concentration and Fe(III) reductase activity. Continued growth of split-root plants in the +Fe/-Fe regime increased the reduction rates of the +Fe-grown portion of the root system over the rates in iron sufficient plants with non-divided roots. The results are discussed in relation to putative factors mediating intra- and interorgan regulation of iron nutrition.     

Reduction of root iron in Plantago lanceolata during recovery from Fe deficiency

Changes in root-associated Fe(III) reductase activity and Fe concentration during recovery from temporary iron starvation were investigated in hydroponically grown Plantago lanceolata L. Within two days, interruption of the Fe supply resulted in enhanced rates of reduction by intact plant roots. Transfer of iron deficient plants to a solution containing various amounts of chelated Fe caused a transient increase in reduction activity before the rates declined to a level determined by the amount of Fe added. Repression of root-associated redox activity was independent of the Fe concentration in the preculture. When iron deficient plants were submitted to a supply of Fe localized to a part of the root system (split-root plants), the decrease in reduction rates was much more pronounced in the Fe-deprived portion of the roots than in the Fe-supplied one. No correlation was observed between root Fe concentration and Fe(III) reductase activity. Continued growth of split-root plants in the +Fe/-Fe regime increased the reduction rates of the +Fe-grown portion of the root system over the rates in iron sufficient plants with non-divided roots. The results are discussed in relation to putative factors mediating intra- and interorgan regulation of iron nutrition.     

Iron reductase systems on the plant plasma membrane—A review

Higher plant roots, leaf mesophyll tissue, protoplasts as well as green algae are able to reduce extra-cellular ferricyanide and ferric chelates. In roots of dicotyledonous and nongraminaceous, monocotyledonous plants, the rate of ferric reduction is increased by iron deficiency. This reduction is an obligatory prerequisite for iron uptake and is mediated by redox systems localized on the plasma membrane. Plasma membrane-bound iron reductase systems catalyze the transmembrane electron transport from cytosolic reduced pyridine nucleotides to extracellular iron compounds. Natural and synthetic ferric complexes can act as electron acceptors.This paper gives an overview about the present knowledge on iron reductase systems at the plant plasma membrane with special emphasis on biochemical characteristics and localisation.     

Impact of iron toxicity on oxidative metabolism in young Eugenia uniflora L. plants

In excess, iron can induce the production and accumulation of reactive oxygen species (ROS), causing oxidative stress. The objective of this work was to evaluate the impact of toxic concentrations of iron (Fe) on the antioxidative metabolism of young Eugenia uniflora plants. Forty-five-day-old plants grown in Hoagland nutrient solution, pH 5.0, were treated with three Fe concentrations, in the form of FeEDTA, during three periods of time. At the end of the treatment, the plants were harvested and relative growth rate, iron content, lipid peroxidation and enzymes and metabolites of the antioxidative metabolism were determined. Iron-treated plants showed higher iron contents, reduced relative growth rates and iron toxicity symptoms in both leaves and roots. There was an increase in lipid peroxidation with increasing Fe, only in the leaves. The enzymatic activities of superoxide dismutase (SOD) and glutathione reductase (GR) increased with increasing Fe concentration and treatment exposure time. The activities of catalase (CAT), peroxidase (POX) and ascorbate peroxidase (APX) also increased with increasing Fe concentration but decreased with increasing treatment exposure time. Glutathione peroxidase activity (GPX) decreased with increasing Fe concentration and exposure time. The ascorbate (AA) and reduced glutathione (GSH) contents and the AA/DHA and GSH/GSSG ratios, in general, increased with increasing Fe concentration and treatment exposure time. The results indicate that under toxic levels of Fe, young E. uniflora plants suffer increased oxidative stress, which is ameliorated through changes in the activities of antioxidative enzymes and in the contents of the antioxidants AA and GSH.     

Influence of chromium(lll) on root-associated Fe(lll) reductase in Plantago lanceolata L.

The application of low chromium concentrations (5 M CrCI₃)decreased chlorophyll content of iron-deficient plants, butdid not cause visual toxicity symptoms in iron-sufficient plants.No significant plant growth differentials were obtained in responseto the various treatments. Chromium application to iron-deficientPlantago lanceolata L. roots increased the activity of root-associatedFe(lll) reductase. This effect was evident only with acceptorsof the turbo reductase and was not observed in iron-sufficientplants. Chromium accumulated in the roots, but was poorly transportedto the leaves. In split-root experiments, which allowed onlya part of the root system to receive chromium, while the otherportion was grown in iron-free medium, roots subjected to eithertreatment showed an intermediate FeEDTA reductase activity withrespect to non-split control plants. It is concluded that amobile factor from the leaves modulates the degree of expressionof the iron stress response. Possible mechanisms by which chromiumaffects Fe metabolism are discussed. Key words: Plantago lanceolata, ferric iron reduction, chromium, iron nutrition, shoot-to-root communication     

PHYTOPLANKTON PLASMA MEMBRANE REDOX ACTIVITY: EFFECT OF IRON LIMITATION AND INTERACTION WITH PHOTOSYNTHESIS1

Phytoplankton plasma membrane electron transport activity was determined by monitoring the reduction of the impermeant artificial electron acceptor ferricyanide in a range of diatoms. The results revealed that constitutive plasma membrane electron transport activity of marine diatoms is high compared with chlorophytes and higher plant cells. Diatom plasma membrane electron transport activity was not significantly increased by iron limitation. This lack of induction on iron limitation indicates that diatoms have an iron acquisition strategy that is distinct from chlorophytes and the dicotyledon higher plants that exhibit marked increases in plasma membrane ferricyanide reductase activity on iron limitation. The interaction of the constitutive plasma membrane electron transport with photosynthesis was also investigated. We found that 1) ferricyanide reduction at the plasma membrane was progressively inhibited in response to increasing irradiances; 2) the presence of extracellular ferricyanide, but not the reduced couple ferrocyanide, caused a marked inhibition of carbon fixation at high irradiance; and 3) extracellular electron acceptors ferricyanide and hexachloroiridate (but not ferrocyanide) induced an immediate and reversible decrease in fluorescence yields (F_o and F_m). The extent to which extracellular electron acceptors affected CO₂ fixation, F_o, and F_m was related to the level of constitutive ferricyanide reductase activity, the species with highest ferricyanide reduction rates being most sensitive. The data suggest that consumption of electrons and/or reductant at the plasma membrane by external acceptors may compete directly with CO₂ fixation for electrons, alter cytosolic‐chloroplast redox poise, and/or induce a redox‐signaling cascade that alters photosynthetic metabolism.     

Plants fabricate Fe-nanocomplexes at root surface to counter and phytostabilize excess ionic Fe

While evaluating the impact of iron nanoparticles (NPs) on terrestrial plants we realized potential of root system of intact plants to form orange–brown complexes constituted of NPs around their roots and at bottom/side of tubes when exposed to FeCl₃. These orange–brown complexes/plaques seen around roots were similar to that reported in wetland plants under iron toxicity. Transmission electron microscopy coupled with energy dispersive X-ray analysis revealed that orange–brown complexes/plaques, formed by root system of all 16 plant species from 11 distinct families tested, were constituted of NPs containing Fe. Selected area electron diffraction and powder X-ray diffraction spectra showed their amorphous nature. Thermogravimetric and fourier transform infra-red analysis showed that these Fe-NPs/nanocomplexes were composed of iron-oxyhydroxide. These plant species generated orange–brown Fe-NPs/nanocomplexes even under strict sterile conditions establishing inbuilt and independent potential of their root system to generate Fe-NPs. Root system of intact plants showed ferric chelate reductase activity responsible for reduction of Fe³⁺ to Fe²⁺. Reduction of potassium ferricyanide by root system of intact plants confirmed that root surface possess strong reducing strength, which could have played critical role in reduction of Fe³⁺ and formation of Fe-NPs/nanocomplexes. Atomic absorption spectrophotometric analysis revealed that majority of iron was retained in Fe-nanocomplexes/plaques, while only 2–3 % was transferred to shoots, indicating formation of nanocomplexes is a phytostabilization mechanism evolved by plants to restrict uptake of iron above threshold levels. We believe that formation of Fe-NPs/nanocomplexes is an ideal homeostasis mechanism evolved by plants to modulate uptake of desired levels of ionic Fe.     

Iron uptake by leaf mesophyll cells: The role of the plasma membrane-bound ferric-chelate reductase

The uptake of ⁵⁹Fe from FeCl₃, ferric (Fe³⁺) citrate (FeCitr) and Fe³⁺-EDTA (FeEDTA) was studied in leaf mesophyll of Vigna unguiculata (L.) Walp. Uptake rates decreased in the order FeCl₃>FeCitrFeEDTA, and uptake depended on an obligatory reduction step of Fe³⁺ to Fe²⁺, after which the ion could be taken up independently of the chelator, citrate. Uptake was strongly increased by photosynthetically active light (>630 nm), and kinetic analysis revealed saturation kinetics with a K _m (FeCitr) of 80–110 M. In the presence of an external Fe²⁺ scavenger, bathophenanthroline disulfonate, the mesophyll also reduced external FeCitr with a K _m of approx. 50–60 M. The reduction rates for FeCitr were five-to eightfold higher than necessary for uptake. Purified plasma membranes from leaves revealed an NADH-dependent FeCitr- and FeEDTA-reductase activity, which had a pH optimum of 6.5–6.8 and a K _m of approx. 20 M for NADH. Under anaerobic conditions, a K _m of 130–170 M for ferric chelates was obtained, while in the presence of oxygen a K _m (FeCitr) of approx. 100 M was found. It is concluded that the leaf plasma membrane provides a ferric-chelate-reductase activity, which plays a crucial role in iron uptake of leaf cells. Under in-vivo conditions, however, reactive oxygen species or strong (blue) light may also contribute to the obligatory reduction of Fe³⁺ prior to uptake.Abbreviations BPDS bathophenanthroline disulfonate - DCMU 3-(3,4 dichlorophenyl)-1,1-dimethyl urea - FCR ferricchelate reductase - FeCitr Fe³⁺-citrate - FeEDTA Fe³⁺-EDTA - PM plasma membrane This work was supported by the SCIENCE program of the European Community (contract no. SC1000344; P.R.M.). We wish to thank P. Siersma and C. Winter for their cooperation at the Central Isotope Laboratory of the Biological Centre of the University of Groningen.     

Water-extractable humic substances enhance iron deficiency responses by Fe-deficient cucumber plants

The ability of Fe-deficient cucumber plants to use iron complexed to a water-extractable humic substances fraction (WEHS), was investigated. Seven-day-old Fe-deficient plants were transferred to a nutrient solution supplemented daily for 5 days with 0.2 μM Fe as Fe-WEHS (5 μg org. C mL^-1), Fe-EDTA, Fe-citrate or FeCl₃. These treatments all allowed re-greening of the leaf tissue, and partial recovery of dry matter accumulation, chlorophyll and iron contents. However, the recovery was faster in plants supplied with Fe-WEHS and was already evident 48 h after Fe supply. The addition of 0.2 μM Fe to the nutrient solution caused also a partial recovery of the dry matter and iron accumulation in roots of Fe-deficient cucumber plants, particularly in those supplied with Fe-WEHS. The addition of WEHS alone (5 μg org. C mL^-1, 0.04 μM Fe) to the nutrient solution slightly but significantly increased iron and chlorophyll contents in leaves of Fe-deficient plants; in these plants, dry matter accumulation in leaves and roots was comparable or even higher than that measured in plants treated with Fe-citrate or FeCl₃. After addition of the different iron sources for 5 days to Fe-deficient roots, morphological modifications (proliferation of lateral roots, increase in the diameter of the sub-apical zones and amplified root-hair formation) and physiological responses (enhanced Fe(III)-chelate reductase and acidification of the nutrient solution) induced by Fe deficiency, were still evident, particularly in plants treated with the humic molecules. The presence of WEHS caused also a further acidification of the nutrient medium by Fe-deficient plants. The Fe-WEHS complex (1 μM Fe) could be reduced by intact cucumber roots, at rates of reduction higher than those measured for Fe-EDTA at equimolar iron concentration. Plasma membrane vesicles, purified by two-phase partition from root microsomes of Fe-deficient plants, were also able to reduce Fe-WEHS. Results show that Fe-deficient cucumber plants can use iron complexed to water soluble humic substances, at least in part via reduction of complexed Fe(III) by the plasma membrane Fe(III)-chelate reductase of root cells. In addition, the stimulating effect of humic substances on H⁺ release might be of relevance for the overall response of the plants to iron shortage. This revised version was published online in June 2006 with corrections to the Cover Date.     

Depolarization of Cell Membrane Potential during Trans-Plasma Membrane Electron Transfer to Extracellular Electron Acceptors in Iron-Deficient Roots of Phaseolus vulgaris L

Transfer of electrons from the cytosol of bean (Phaseolus vulgaris L.) root cells to extracellular acceptors such as ferricyanide and Fe^IIIEDTA causes a rapid depolarization of the membrane potential. This effect is most pronounced (30-40 millivolts) with root cells of Fe-deficient plants, which have an increased capacity to reduce extracellular ferric salts. Ferrocyanide has no effect. In the state of ferricyanide reduction, H⁺ (1H⁺/2 electrons) and K⁺ ions are excreted. The reduction of extracellular ferric salts by roots of Fe-deficient bean plants is driven by cellular NADPH (Sijmons, van den Briel, Bienfait 1984 Plant Physiol 75: 219-221). From this and from the membrane potential depolarization, we conclude that trans-plasma membrane electron transfer from NADPH is the primary process in the reduction of extracellular ferric salts.     

Sites of absorption and translocation of iron in barley roots: tracer and microautoradiographic studies

Absorption and translocation of labeled Fe were measured at various locations along the length of intact seminal axes and lateral roots of iron-sufficient (+Fe) and iron-stressed (−Fe) barley (Hordeum vulgare) plants. In seminal axes of +Fe plants, rates of translocation were very much higher in a zone 1 to 4 cm from the root tip than elsewhere in the root. Lateral roots of high rates of translocation were also restricted to a narrow band of maturing or recently matured cells. In −Fe plants the patterns of uptake and translocation were essentially the same as in +Fe plants but the rates were 7- to 10-fold higher. The amount of labeled Fe bound to the root itself was not increased by Fe stress and its distribution along the root seemed inversely related to the ability to translocate Fe.

Microautoradiographic studies showed that most of the iron bound to roots was held in an extracellular peripheral band in which iron seemed to be precipitated. This process may be assisted by microbial colonies but did not depend on them since it was seen, although to a lesser extent, in sterile roots. In zones from which iron was translocated there was evidence that internal root tissues became labeled readily, but as translocation declined with distance from the root tip, radial penetration of Fe appeared to become progressively less. The results are discussed in relation to possible changes in the pH or redox potential of the surface of the root.

     

The induction of the “turbo reductase” is inhibited by cycloheximide, cordycepin and ethylene inhibitors in Fe-deficient cucumber (Cucumis sativus L.) plants

Summary Dicotyledonous plants respond to Fe deficiency by enhancing the capacity of their roots to reduce Fe(III) to Fe(II). It has been suggested that there are two different ferric redox systems in the roots: the standard reductase, active with ferricyanide and not inducible by Fe deficiency, and the turbo reductase, active with both ferricyanide and ferric chelates and inducible by Fe deficiency. We have used different experimental approaches to test whether or not the Fe(III)-reducing capacity of cucumber (Cucumis sativus L. cv. Ashley) roots can be explained by considering the standard and the turbo reductase as the same enzyme. For this, we used both Fe-sufficient and Fe-deficient plants, which were treated with ethylene inhibitors (cobalt or silver thiosulfate; found to inhibit the turbo reductase in a previous work), a protein synthesis inhibitor (cycloheximide), or an mRNA polyadenylation inhibitor (cordycepin). At different times after application of these inhibitors, reduction of both ferricyanide and Fe(III)-EDTA were determined. In addition, we studied the effects of pH and temperature on the reduction of ferricyanide and Fe(III)-EDTA by both Fe-sufficient and Fe-deficient plants. Results suggest that there are, at least, two different ferric redox systems in the roots. Enhancement of Fe(III)-reducing capacity (turbo reductase) by Fe-deficient plants probably requires the de novo synthesis of a (or several) protein(s), which has a high turnover rate and whose expression is presumably regulated by ethylene.Abbreviations Ch-R ferric chelate reductase - CHM cycloheximide - CN-R ferricyanide reductase - EDDHA N,N-ethylene bis[2-(2-hydroxyphenyl)-glycine] - EDTA ethylenediamine-tetraacetic acid - Ferrozine 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine - HEDTA N-hydroxyethylethylene-diaminetriacetic acid - STS silver thiosulfate     

Effect of primary leaves on 59Fe uptake by roots and 59Fe distribution in the shoot of iron sufficient and iron deficient bean (Phaseolus vulgaris L.) plants

A study has been made on the effect of primary leaves on iron (Fe) distribution in the shoot. Bean (Phaseolus vulgaris L.) seedlings were precultured in nutrient solution with 8×10^-5 M FeEDTA for 4 days, and then grown further with either 8×10^-5 M FeEDTA (+Fe) or without Fe supply (-Fe) for another 5 days. Thereafter, both +Fe and -Fe plants were treated in three different ways: undisturbed; one primary leaf removed; or one primary leaf shaded, starting two hours before supply ⁵⁹FeEDTA to the roots. The +Fe plants were supplied with 8×10^-5 M ⁵⁹FeEDTA, and the -Fe plants with only 1×10^-6 M ⁵⁹FeEDTA. After 1 to 8 hour uptake periods, plants were harvested and ⁵⁹Fe in different organs was determined. Removal or shading of one primary leaf did not affect ⁵⁹Fe uptake by roots and ⁵⁹Fe translocation to the shoot in +Fe plants. In the -Fe plants, however, removal of one primary leaf decreased ⁵⁹Fe uptake by roots, whereas shading of one primary leaf had no effect on ⁵⁹Fe uptake but slightly enhanced ⁵⁹Fe translocation from roots to the shoot. The quantity of ⁵⁹Fe in primary leaves was positively correlated with quantity of ⁵⁹Fe in the stem in the -Fepplants, but not in the +Fe plants. In both, the +Fe and -Fe plants, the quantity of ⁵⁹Fe in the shoot apex was positively correlated with ⁵⁹Fe in primary leaves. The results suggest that irrespective of the Fe nutritional status of plants, the source of Fe for the shoot apex is Fe retranslocated from primary leaves.     

Evidence for electron transport across the plasma membrane of Zea mays root cells

Exogenous ferricyanide is reduced by roots of Z. mays. In contrast to oxidation of exogenous electron donors, ferricyanide reduction occurs mostly at the apical 5 mm of the root. Using just this portion of the root, it is shown that the activity is neither a consequence of uptake of ferricyanide followed by excretion of its reduced form, nor of leakage of a reductant. Addition of ferricyanide for 40 s or 5 min results in an apparent oxidation of NADPH but not of NADH; rates of ferricyanide reduction vary together with levels of NADPH but not of NADH in the presence or absence of oxygen. It is concluded that an enzyme which can oxidize cytoplasmic NADPH and transfer the electrons to an external acceptor exists at the cell surface of maize roots. This finding extends the results of others who showed similar redox activity at the surface of Fe-depleted dicotyledonous roots, and indicates that an energy source other than ATP exists at the cell surface of a variety of plants under unstressed conditions.     

Iron-Stress Induced Redox Activity in Tomato (Lycopersicum esculentum Mill.) Is Localized on the Plasma Membrane

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 Fe-sufficient 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.     

The relationship between the activity of various iron-containing and iron-free enzymes and the presence of nicotianamine in tomato seedlings

The influence of nicotianamine (NA) and iron on the activities of 4 iron-containing and two iron-free enzymes in leaves and roots of the NA-free tomato mutant chloronerva and its NA-containing wild-type ( Lycopersicon esculentum Mill. cv. Bonner Beste) was investigated. Aconitase (EC 4.2.1.3) activity in both leaves and roots was much higher in the mutant under normal iron supply (10 μ M FeEDTA) and in wild-type under iron deficiency than in wild-type supplied with 10 μ M FeEDTA. Application of NA to chloronerva leaves led to a decrease of aconitase activity in leaves and roots. NA had no effect on the enzyme activity when added to the assay medium.
Similar results were obtained for the iron-containing enzymes catalase (EC 1.11.1.6), ascorbate-dependent peroxidase (EC 1.11.1.11) and guaiacol-dependent peroxidase (EC 1.11.1.7) in roots. NA treatment of the mutant leaves decreased enzyme activities in roots down to wild-type values. In vivo NA application had no effect on enzyme activities in leaf extracts.
The activities of the iron-free enzymes NAD⁺-malate dehydrogenase (EC 1.1.1.37) and phosphofructokinase (EC 2.7.1.11) in root and leaf extracts were not influenced by the iron supply to the plants.