Does Iron Deficiency in Pisum sativum Enhance the Activity of the Root Plasmalemma Iron Transport Protein?

Roots of Fe-sufficient and Fe-Deficient pea (Pisum sativum L.) were studied to determine the effect of Fe-deficiency on the activity of the root-cell plasmalemma Fe²⁺ transport protein. Rates of Fe(III) reduction and short-term Fe²⁺ influx were sequentially determined in excised primary lateral roots using Fe(III)-ethylene-diaminetetraacetic acid (Fe[III]-EDTA). Since the extracellular Fe²⁺ for membrane transport was generated by root Fe(III) reduction, rates of Fe²⁺ influx for each root system were normalized on the basis of Fe(III) reducing activity. Ratios of Fe²⁺ influx to Fe(III) reduction (micromole Fe²⁺ absorbed/micromole Fe[III] reduced) revealed no enhanced Fe²⁺ transport capacity in roots of Fe-deficient peas (from the parental genotype, Sparkle) or the functional Fe-deficiency pea mutant, E107 (derived from Sparkle), relative to roots of Fe-sufficient Sparkle plants. Data from studies using 30 to 100 micromolar Fe(III)-EDTA indicated a linear relationship between Fe²⁺ influx and Fe(III) reduction (Fe²⁺ generation), while Fe²⁺ influx saturated at higher concentrations of Fe(III)-EDTA. Estimations based on current data suggest the Fe²⁺ transport protein may saturate in the range of 10^−4.8 to 10⁻⁴ molar Fe²⁺. These results imply that for peas, the physiological rate limitation to Fe acquisition in most well-aerated soils would be the root system's ability to reduce soluble Fe(III)-compounds.     

SPATIAL AND TEMPORAL DEVELOPMENT OF IRON(III) REDUCTASE ACTIVITY IN ROOT SYSTEMS OF PISUM SATIVUM (FABACEAE) CHALLENGED WITH IRON-DEFICIENCY STRESS

The development of plasma membrane-associated iron(III) reductase activity was characterized in root systems of Pisum sativum during the first 2 wk of growth, as plants were challenged with iron-deficiency stress. Plants of a parental genotype (cv. Sparkle) and a functional iron-deficiency mutant genotype (E107) were grown hydroponically with or without supplemental iron. Iron(III) reductase activity was visualized by placing the roots in an agarose matrix containing 0.2 idm Fe(III)-ethylenediaminetetraacetic acid and 0.3 mM Na₂-bathophenanthrolinedisulfonic acid (BPDS). Red staining patterns, resulting from the formation of Fe(II)-BPDS, were used to identify iron(III)-reducing regions. Iron(III) reduction was extensive on roots of E107 as early as d 7, but not until d 11 for -Fe-treated Sparkle. Roots of +Fe-treated Sparkle showed limited regions of reductase activity throughout the period of study. For secondary lateral roots, iron(III) reduction was found for all growth types except + Fe-treated Sparkle. Treating Sparkle plants alternately to a cycle of iron deficiency, iron sufficiency, and iron deficiency revealed that reductase activity at a given root zone could be alternatively present, absent, and again present. Our results suggest that for Pisum roots grown under the present conditions, iron-deficiency stress induces the activation of iron(III) reductase capacity within 2 d.     

豌豆Sparkle及其单基因突变体E107的铁锰吸收及转移效率

用豌豆Ｓｐａｒｋｌｅ及其单基因突变体Ｅ１０７进行的水培的试验表明,－Ｆｅ和＋Ｆｅ处理的Ｅ１０７幼苗以及－Ｆｅ处理的Ｓｐａｒｋｌｅ幼苗均表现出根系Ｈ＋分泌量大、根系Ｆｅ（Ⅲ）还原力强等特点,其中尤以＋Ｆｅ处理的Ｅ１０７最为突出;而十Ｆｅ处理的Ｓｐａｒｋｌｅ则无以上特点。与Ｓｐａｒｋｌｅ相比,Ｅ１０７各处理的地上部Ｆｅ、Ｍｎ合量均很高,但根部含量则相反。与Ｓｐｅｋｌｅ相比,Ｅ１０７—Ｆｅ处理表现为Ｆｅ高效,即使在＋Ｆｅ处理下,Ｅ１０７仍表现出－Ｆｅ条件下的根系生理特性,活化并还原了根际大量Ｆｅ（Ⅲ）和Ｍｎ,因而它对Ｆｅ、Ｍｎ具有较高的吸收效率,但是这些元素并不在根系中贮存,而是源源不断地运输到地上部,并在叶片中累积乃至使叶片中毒坏死,充分表现了Ｅ１０７单基因突变体对Ｆｅ、Ｍｎ也具有较高的转移效率。     

Physiological Characteristics of Fe Accumulation in the ;Bronze' Mutant of Pisum sativum L., cv ;Sparkle' E107 (brz brz)

The pea (Pisum sativum L.) mutant, E107 (brz, brz) accumulated extremely high concentrations of Fe in its older leaves when grown in light rooms in either defined nutrient media or potting mix, or outdoors in soil. Leaf symptoms (bronze color and necrosis) were correlated with very high Fe concentrations. When E107 plants were grown in nutrient solutions supplied 10 μm Fe, as the Fe(III)-N,N′-ethylenebis[2-(2-hydroxyphenyl)glycine] chelate, their roots released higher concentrations of Fe(III) reducing substances to the nutrient media than did roots of the normal parent cv, `Sparkle.' Reciprocal grafting experiments demonstrated that the high concentrations of Fe in the shoot was controlled by the genotype of the root. In short-term <sup>59</sup>Fe uptake studies, 15-day-old E107 seedlings exhibited higher rates of Fe absorption than did `Sparkle' seedlings under Fe-adequate growth conditions. Iron deficiency induced accelerated short-term Fe absorption rates in both mutant and normal genotypes. Iron-treated E107 roots also released larger amounts of both protons and Fe(III) reductants into their nutrient media than did iron-treated `Sparkle' roots. Furthermore, the mutant translocated proportionately more Fe to its shoot than did the parent regardless of Fe status.     

Shoot-to-Root Signal Transmission Regulates Root Fe(III) Reductase Activity in the dgl Mutant of Pea

To understand the root, shoot, and Fe-nutritional factors that regulate root Fe-acquisition processes in dicotyledonous plants, Fe(III) reduction and net proton efflux were quantified in root systems of an Fe-hyperaccumulating mutant (dgl) and a parental (cv Dippes Gelbe Viktoria [DGV]) genotype of pea (Pisum sativum). Plants were grown with (+Fe treated) or without (-Fe treated) added Fe(III)-N,N'-ethylenebis[2-(2-hydroxyphenyl)-glycine] (2 [mu]M); root Fe(III) reduction was measured in solutions containing growth nutrients, 0.1 mM Fe(III)-ethylenediaminetetraacetic acid, and 0.1 mM Na2-bathophenanthrolinedisulfonic acid. Daily measurements of Fe(III) reduction (d 10-20) revealed initially low rates in +Fe-treated and -Fe-treated dgl, followed by a nearly 5-fold stimulation in rates by d 15 for both growth types. In DGV, root Fe(III) reductase activity increased only minimally by d 20 in +Fe-treated plants and about 3-fold in -Fe-treated plants, beginning on d 15. Net proton efflux was enhanced in roots of -Fe-treated DGV and both dgl growth types, relative to +Fe-treated DGV. In dgl, the enhanced proton efflux occurred prior to the increase in root Fe(III) reductase activity. Reductase studies using plants with reciprocal shoot:root grafts demonstrated that shoot expression of the dgl gene leads to the generation of a transmissible signal that enhances Fe(III) reductase activity in roots. The dgl gene product may alter or interfere with a normal component of a signal transduction mechanism regulating Fe homeostasis in plants.     

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 Role of Iron-Deficiency Stress Responses in Stimulating Heavy-Metal Transport in Plants

Plant accumulation of Fe and other metals can be enhanced under Fe deficiency. We investigated the influence of Fe status on heavy-metal and divalent-cation uptake in roots of pea (Pisum sativum L. cv Sparkle) seedlings using Cd²⁺ uptake as a model system. Radiotracer techniques were used to quantify unidirectional ¹⁰⁹Cd influx into roots of Fe-deficient and Fe-sufficient pea seedlings. The concentration-dependent kinetics for ¹⁰⁹Cd influx were graphically complex and nonsaturating but could be resolved into a linear component and a saturable component exhibiting Michaelis-Menten kinetics. We demonstrated that the linear component was apoplastically bound Cd²⁺ remaining in the root cell wall after desorption, whereas the saturable component was transporter-mediated Cd²⁺ influx across the root-cell plasma membrane. The Cd²⁺ transport system in roots of both Fe-deficient and Fe-sufficient seedlings exhibited similar Michaelis constant values, 1.5 and 0.6 μm, respectively, for saturable Cd²⁺ influx, whereas the maximum initial velocity for Cd²⁺ uptake in Fe-deficient seedlings was nearly 7-fold higher than that in Fe-grown seedlings. Investigations into the mechanistic basis for this response demonstrated that Fe-deficiency-induced stimulation of the plasma membrane H⁺-ATPase did not play a role in the enhanced Cd²⁺ uptake. Expression studies with the Fe²⁺ transporter cloned from Arabidopsis, IRT1, indicated that Fe deficiency induced the expression of this transporter, which might facilitate the transport of heavy-metal divalent cations such as Cd²⁺ and Zn²⁺, in addition to Fe²⁺.     

Formate Dehydrogenase, an Enzyme of Anaerobic Metabolism, Is Induced by Iron Deficiency in Barley Roots

To identify the proteins induced by Fe deficiency, we have compared the proteins of Fe-sufficient and Fe-deficient barley (Hordeum vulgare L.) roots by two-dimensional polyacrylamide gel electrophoresis. Peptide sequence analysis of induced proteins revealed that formate dehydrogenase (FDH), adenine phosphoribosyltransferase, and the Ids3 gene product (for Fe deficiency-specific) increased in Fe-deficient roots. FDH enzyme activity was detected in Fe-deficient roots but not in Fe-sufficient roots. A cDNA encoding FDH (Fdh) was cloned and sequenced. Fdh expression was induced by Fe deficiency. Fdh was also expressed under anaerobic stress and its expression was more rapid than that induced by Fe deficiency. Thus, the expression of Fdh observed in Fe-deficient barley roots appeared to be a secondary effect caused by oxygen deficiency in Fe-deficient plants.     

Iron Transport to Developing Ovules of Pisum sativum (I. Seed Import Characteristics and Phloem Iron-Loading Capacity of Source Regions)

To understand the processes that control Fe transport to developing seeds, we have characterized seed growth and Fe accretion and have developed a radiotracer technique for quantifying phloem Fe loading in vegetative source regions of Pisum sativum. In hydroponically grown plants of cv Sparkle, developing ovules exhibited a seed-growth period of 22 d, with Fe import occurring throughout the 22-d period. Average Fe content of mature seeds was 19 [mu]g. Source tissues of intact plants were abraded and pulse labeled for 4 h with 100 [mu]M 59Fe(III)-citrate. Fe was successfully phloem loaded and transported to seeds from leaflets, stipules, and pod walls. Total export of 59Fe from labeled source regions was used to calculate tissue-loading rates of 36, 40, and 51 pmol of Fe cm-2 h-1 for the leaflet, stipule, and pod wall surfaces, respectively. By comparison, surface area measurements, along with seed-growth results, allowed us to calculate average theoretical influx values of 42 or 68 pmol of Fe cm-2 h-1 for vegetative tissues at nodes with one or two pods, respectively. Additional studies with the regulatory pea mutant, E107 (a single-gene mutant of cv Sparkle that can overaccumulate Fe), enabled us to increase Fe delivery endogenously to the vegetative tissues. A 36-fold increase in Fe content of E107 leaves, relative to Sparkle, resulted in no increase in Fe content of E107 seeds. Based on these findings, we hypothesized that Fe is phloem loaded in a chelated form, and the expression/synthesis of the endogenous chelator is an important factor in the control of Fe transport to the seeds.     

Interrelationships between trans-Plasma Membrane Electron/Proton Transfer Stoichiometry, Organic Acid Metabolism, and Nitrate Reduction in Dwarf Bean (Phaseolus vulgaris)

Iron deficiency in dwarf bean (Phaseolus vulgaris L.) induces an increased activity of a system in the rhizodermal cells, which reduces extracellular ferric salts, and an active proton efflux from the roots, which is coupled to accumulation of citrate and malate in the roots and subsequent export of these compounds in the xylem. During reduction of extracellular ferricyanide by Fe-deficient plants, the stoichiometry of electron transport to proton efflux is 2e⁻/1H⁺, and citrate and malate levels in the roots are strongly decreased. Reduction of ferricyanide by Fe-sufficient plants has no influence on root and shoot levels of citrate and malate, but in such plants the process is characterized by a e⁻/H⁺ efflux stoichiometry close to unity. Apparently, organic acid metabolism and transport are closely associated with the e⁻/H⁺ efflux ratio. To assess the significance of organic acid metabolism as one of the direct intracellular components of the induced unbalanced e⁻/H⁺ efflux by roots, we studied NO₃⁻ reduction in shoots and roots of Fe-deficient and Fe-sufficient plants. Nitrate reductase activity in the roots was positively correlated with the level of citrate and malate, whereas the enzyme activity in the leaves responded positively to the import of these organic acid anions.     

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.     

Acetate Threshold Concentrations Suggest Varying Energy Requirements during Anaerobic Respiration by Anaeromyxobacter dehalogenans

Acetate threshold concentrations were determined under chlororespiring and Fe(III)-reducing conditions for Anaeromyxobacter dehalogenans strain 2CP-C. The acetate threshold concentrations measured were 69 ± 4, 19 ± 8, and <1 nM for chlororespiration, amorphous Fe(III) reduction, and Fe(III) citrate reduction, respectively. Residual ΔG values of −75.4 kJ/mol of electrons for chlororespiration and −41.5 kJ/mol of electrons for amorphous Fe(III) reduction were calculated at the acetate threshold concentration. By comparing threshold concentrations for different metabolisms in a single organism, this study provides insight into the metabolic use of energy under different growth conditions.     

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     

Microbial Iron Reduction by Enrichment Cultures Isolated from Estuarine Sediments

Microbial Fe reduction in acetate- and succinate-containing enrichment cultures initiated with an estuarine sediment inoculum was studied. Fe reduction was unaffected when SO₄²⁻ reduction was inhibited by MoO₄²⁻, indicating that both processes could occur independently. Bacterially produced sulfide precipitated as FeS but was not completely responsible for Fe reduction. The separation of oxidized Fe particles from bacteria by dialysis tubing demonstrated that direct bacterial contact was necessary for Fe reduction. Fe reduction in cultures amended with NO₃⁻ was delayed until NO₃⁻ and NO₂⁻ were removed. However, bacterial attachment to oxidized Fe particles in NO₃⁻-amended cultures occurred early during growth in a manner similar to NO₃⁻-free cultures. During late stages of growth, bacteria not attached to Fe particles became pale and swollen, while attached cells remained bright blue when examined by 4′,6-diamidine-2-phenylindole epifluo-rescence microscopy. The presence of added oxidized Mn had no effect on Fe reduction. The results suggested that enzymatic Fe reduction was responsible for reducing Fe in these cultures even in the presence of sulfide and that cells incapable of Fe reduction became unhealthy when Fe(III) was the only available electron acceptor.     

The Impact of Gamma Radiation on Sediment Microbial Processes

Microbial communities have the potential to control the biogeochemical fate of some radionuclides in contaminated land scenarios or in the vicinity of a geological repository for radioactive waste. However, there have been few studies of ionizing radiation effects on microbial communities in sediment systems. Here, acetate and lactate amended sediment microcosms irradiated with gamma radiation at 0.5 or 30 Gy h⁻¹ for 8 weeks all displayed NO₃⁻ and Fe(III) reduction, although the rate of Fe(III) reduction was decreased in 30-Gy h⁻¹ treatments. These systems were dominated by fermentation processes. Pyrosequencing indicated that the 30-Gy h⁻¹ treatment resulted in a community dominated by two Clostridial species. In systems containing no added electron donor, irradiation at either dose rate did not restrict NO₃⁻, Fe(III), or SO₄²⁻ reduction. Rather, Fe(III) reduction was stimulated in the 0.5-Gy h⁻¹-treated systems. In irradiated systems, there was a relative increase in the proportion of bacteria capable of Fe(III) reduction, with Geothrix fermentans and Geobacter sp. identified in the 0.5-Gy h⁻¹ and 30-Gy h⁻¹ treatments, respectively. These results indicate that biogeochemical processes will likely not be restricted by dose rates in such environments, and electron accepting processes may even be stimulated by radiation.     

Chemical and Biological Interactions during Nitrate and Goethite Reduction by Shewanella putrefaciens 200

Although previous research has demonstrated that NO₃⁻ inhibits microbial Fe(III) reduction in laboratory cultures and natural sediments, the mechanisms of this inhibition have not been fully studied in an environmentally relevant medium that utilizes solid-phase, iron oxide minerals as a Fe(III) source. To study the dynamics of Fe and NO₃⁻ biogeochemistry when ferric (hydr)oxides are used as the Fe(III) source, Shewanella putrefaciens 200 was incubated under anoxic conditions in a low-ionic-strength, artificial groundwater medium with various amounts of NO₃⁻ and synthetic, high-surface-area goethite. Results showed that the presence of NO₃⁻ inhibited microbial goethite reduction more severely than it inhibited microbial reduction of the aqueous or microcrystalline sources of Fe(III) used in other studies. More interestingly, the presence of goethite also resulted in a twofold decrease in the rate of NO₃⁻ reduction, a 10-fold decrease in the rate of NO₂⁻ reduction, and a 20-fold increase in the amounts of N₂O produced. Nitrogen stable isotope experiments that utilized δ¹⁵N values of N₂O to distinguish between chemical and biological reduction of NO₂⁻ revealed that the N₂O produced during NO₂⁻ or NO₃⁻ reduction in the presence of goethite was primarily of abiotic origin. These results indicate that concomitant microbial Fe(III) and NO₃⁻ reduction produces NO₂⁻ and Fe(II), which then abiotically react to reduce NO₂⁻ to N₂O with the subsequent oxidation of Fe(II) to Fe(III).     

Characterization of Phloem iron and its possible role in the regulation of fe-efficiency reactions

`Fe-efficiency reactions' are induced in the roots of dicotyledonous plants as a response to Fe deficiency. The role of phloem Fe in the regulation of these reactions was investigated. Iron travels in the phloem of Ricinus communis L. as a complex with an estimated molecular weight of 2400, as determined by gel exclusion chromatography. The complex is predominantly in the ferric form, but because of the presence of reducing compounds in the phloem sap, there must be a fast turnover in situ between ferric and ferrous (k ≈ 1 min⁻¹). Iron concentrations in R. communis phloem were determined colorimetrically or after addition of ⁵⁹Fe to the nutrient solution. The iron content of the phloem in Fe-deficient plants was lower (7 micromolar) than in Fe-sufficient plants (20 micromolar). Administration of Fe-EDTA to leaves of Phaseolus vulgaris L. increased the iron content of the roots within 2 days, and decreased proton extrusion and ferric chelate reduction. The increase in iron content of the roots was about the same as the difference between iron contents of roots grown on two iron levels with a concomitantly different expression of Fe-efficiency reactions. We conclude that the iron content of the leaves is reflected by the iron content of the phloem sap, and that the capacity of the phloem to carry iron to the roots is sufficient to influence the development of Fe-efficiency reactions. This does not preclude other ways for the shoot to influence these reactions.     

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.     

The role of active Bradyrhizobium japonicum in iron stress response of soybeans

The objective of this study was to identify the sites of H-ion exudation and Fe(III) reduction along both inoculated and non-inoculated roots of A7 and T203 soybeans. A split-root system was used in which half the roots of each plant were inoculated and actively fixing nitrogen and the other half were not. Expectedly, the Fe-stress response was strong on both sides of the split-root system in the +N-Fe treatment of variety A7 (inactive nodules) but not of variety T203. The Fe-stress response of A7 was enhanced by the presence of active nodules. Variety T203 is Fe inefficient and normally fails to produce any Fe-stress response, but in the absence of nitrogen and iron (–N–Fe), inoculated roots responded to Fe stress with exudation of both H-ions and reductants. Intact split-root systems were embedded in agar to determine the location of H-ion exudation and Fe(III) reduction. On the inoculated side of the –N–Fe and –N+Fe treatments (active nodules) of both soybean varieties, H-ion production was associated mainly with the active nodules. However, quantities of H-ion release were much greater under Fe stress (–N–Fe) than with adequate Fe (–N+Fe). Reduction of Fe(III) to Fe(II) was found only on the nodulated side with T203, but on both sides with A7. In variety T203 the Fe reduction was associated with younger roots located just below the nodule clusters on the inoculated side of the –N treatments. Active nodules appear to play a key role in the Fe-deficiency stress response of T203 soybean.     

Induction of the Root Cell Plasma Membrane Ferric Reductase (An Exclusive Role for Fe and Cu)

Induction of ferric reductase activity in dicots and nongrass monocots is a well-recognized response to Fe deficiency. Recent evidence has shown that Cu deficiency also induces plasma membrane Fe reduction. In this study we investigated whether other nutrient deficiencies could also induce ferric reductase activity in roots of pea (Pisum sativum L. cv Sparkle) seedlings. Of the nutrient deficiencies tested (K, Mg, Ca, Mn, Zn, Fe, and Cu), only Cu and Fe deficiencies elicited a response. Cu deficiency induced an activity intermediate between Fe-deficient and control plant activities. To ascertain whether the same reductase is induced by Fe and Cu deficiency, concentration- and pH-dependent kinetics of root ferric reduction were compared in plants grown under control, -Fe, and -Cu conditions. Additionally, rhizosphere acidification, another process induced by Fe deficiency, was quantified in pea seedlings grown under the three regimes. Control, Fe-deficient, and Cu-deficient plants exhibited no major differences in pH optima or Km for the kinetics of ferric reduction. However, the Vmax for ferric reduction was dramatically influenced by plant nutrient status, increasing 16- to 38-fold under Fe deficiency and 1.5- to 4-fold under Cu deficiency, compared with that of control plants. These results are consistent with a model in which varying amounts of the same enzyme are deployed on the plasma membrane in response to plant Fe or Cu status. Rhizosphere acidification rates in the Cu-deficient plants were similarly intermediate between those of the control and Fe-deficient plants. These results suggest that Cu deficiency induces the same responses induced by Fe deficiency in peas.