Characterization of FRO1, a pea ferric-chelate reductase involved in root iron acquisition

To acquire iron, many plant species reduce soil Fe(III) to Fe(II) by Fe(III)-chelate reductases embedded in the plasma membrane of root epidermal cells. The reduced product is then taken up by Fe(II) transporter proteins. These activities are induced under Fe deficiency. We describe here the FRO1 gene from pea (Pisum sativum), which encodes an Fe(III)-chelate reductase. Consistent with this proposed role, FRO1 shows similarity to other oxidoreductase proteins, and expression of FRO1 in yeast conferred increased Fe(III)-chelate reductase activity. Furthermore, FRO1 mRNA levels in plants correlated with Fe(III)-chelate reductase activity. Sites of FRO1 expression in roots, leaves, and nodules were determined. FRO1 mRNA was detected throughout the root, but was most abundant in the outer epidermal cells. Expression was detected in mesophyll cells in leaves. In root nodules, mRNA was detected in the infection zone and nitrogen-fixing region. These results indicate that FRO1 acts in root Fe uptake and they suggest a role in Fe distribution throughout the plant. Characterization of FRO1 has also provided new insights into the regulation of Fe uptake. FRO1 expression and reductase activity was detected only in Fe-deficient roots of Sparkle, whereas both were constitutive in brz and dgl, two mutants with incorrectly regulated Fe accumulation. In contrast, FRO1 expression was responsive to Fe status in shoots of all three plant lines. These results indicate differential regulation of FRO1 in roots and shoots, and improper FRO1 regulation in response to a shoot-derived signal of iron status in the roots of the brz and dgl mutants.     

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.     

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。     

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.     

Physiological Characterization of a Single-Gene Mutant of Pisum sativum Exhibiting Excess Iron Accumulation: I. Root Iron Reduction and Iron Uptake

Root systems of mutant (E107) and parental (cv `Sparkle') Pisum sativum genotypes were studied to determine the basis for excess Fe accumulation in E107. Plants were grown with (+Fe-treated) or without (−Fe-treated) added Fe(III)-N,N'-ethylenebis[2-(2-hydroxyphenyl)glycine] in aerated nutrient solutions. Daily measurements of Fe(III) reduction indicated a four-to seven-fold higher reduction rate in +Fe- or −Fe-treated E107, and −Fe-treated Sparkle, when compared with +Fe-treated Sparkle. An agarose-based staining technique used to localize Fe(III) reduction, revealed Fe(III) reduction over most of the length of the roots (but not at the root apices) in both E107 treatments and −Fe-treated Sparkle. In +Fe-treated Sparkle, Fe(III) reduction was either nonexistent or localized to central regions of the roots. Measurements of short-term Fe influx (with 0.1 millimolar ⁵⁹Fe(III)-ethylenediaminetetraacetic acid) was also enhanced (threefold) in +Fe- or −Fe-treated E107 and −Fe-treated Sparkle, relative to +Fe-treated Sparkle. The physiological characteristics of E107 root systems, which are similar to those seen in Fe-deficient Sparkle, have led us to conclude that the mutation causes E107 to act functionally as an Fe-deficient plant, and appears to explain the excess Fe accumulation in E107.     

Glyphosate inhibition of ferric reductase activity in iron deficient sunflower roots

Iron (Fe) deficiency is increasingly being observed in cropping systems with frequent glyphosate applications. A likely reason for this is that glyphosate interferes with root uptake of Fe by inhibiting ferric reductase in roots required for Fe acquisition by dicot and nongrass species. This study investigated the role of drift rates of glyphosate (0.32, 0.95 or 1.89 mm glyphosate corresponding to 1, 3 and 6% of the recommended herbicidal dose, respectively) on ferric reductase activity of sunflower (Helianthus annuus) roots grown under Fe deficiency conditions. Application of 1.89 mm glyphosate resulted in almost 50% inhibition of ferric reductase within 6 h and complete inhibition 24 h after the treatment. Even at lower rates of glyphosate (e.g. 0.32 mm and 0.95 mm), ferric reductase was inhibited. Soluble sugar concentration and the NAD(P)H oxidizing capacity of apical roots were not decreased by the glyphosate applications. To our knowledge, this is the first study reporting the effects of glyphosate on ferric reductase activity. The nature of the inhibitory effect of glyphosate on ferric reductase could not be identified. Impaired ferric reductase could be a major reason for the increasingly observed Fe deficiency in cropping systems associated with widespread glyphosate usage.     

Is ethylene involved in regulation of root ferric reductase activity of dicotyledonous species under iron deficiency?

Most dicotyledonous species respond to Fe deficiency by developing some mechanisms known as Fe-deficiency responses. The role of ethylene in regulation of root ferric reductase activity of wild-type tomato (Lycopersicon esculentum L.) and its mutant Never ripe (Nr), bean (Phaseolus vulgaris L., cv. Bifeng 80-30), and cucumber (Cucumis sativus L., cv. Xintaimici) plants grown in nutrient solution without Fe supply was studied under controlled condition. The results show that: (i) the tomato mutant Nr, which is insensitive to ethylene, presented rapid increase in root ferric reductase activity after omitting Fe from the nutrient solution; (ii) the initial time for increase in root ferric reductase activity was earlier than that in ethylene production after onset of Fe deficiency in the three species; (iii) like cobalt (3 μM Co²⁺), an inhibitor for ethylene production, high concentration of zinc (50 μM Zn²⁺) and copper (5 μM Cu²⁺) also suppressed the increase in root ferric reductase activity of Fe-starved plants; (iv) under Fe-sufficient conditions, indol-3-butylric acid (IBA) stimulated root ferric reductase activity of cucumber and bean plants, and this stimulating effect could not be suppressed by aminoethoxyvinylglycine (AVG, an inhibitor for ethylene synthesis). These results suggested that ethylene might not be directly involved in the regulation of root ferric reductase activity of Fe-deficient dicotyledonous species.     

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.     

The Iron-Deficiency Induced Phenolics Accumulation May Involve in Regulation of Fe(III) Chelate Reductase in Red Clover

Although considerable researches have been conducted on the physiological responses to plant iron (Fe) deficiency stress in dicotyledonous plants, much still needs to be learned about the regulation of these processes. In the present research, red clover was used to investigate the role of root phenolics accumulation in regulating Fe-deficiency induced Fe(III) chelate reductase (FCR). The root FCR activity, IAA and phenolics accumulation, and also the phenolics secretion were greatly increased by the Fe deficiency treatment. The application of TIBA (2,3,5-triiodobenoic acid) to the stem, an IAA polar transport inhibitor, which could decrease IAA accumulation in root, significantly inhibited the FCR activity, but did not effect root phenolics accumulation and secretion, suggesting that IAA itself did not involve in root phenolics accumulation and secretion. In contrast, the Fe deficiency treatment significantly decreased the root IAA-oxidase activity. Interestingly the phenolics extracted from roots inhibited IAA-oxidase activity in vitro, and this inhibition was greater with phenolics extracted from roots of Fe deficient plants than that from Fe sufficient plants, indicating that the Fe deficiency-induced IAA-oxidase inhibition probably caused by the phenolics accumulation in Fe deficient roots. Based on these observations, we propose a model where under Fe deficiency stress in dicots, an increase in root phenolics concentrations plays a role in regulating root IAA levels through an inhibition of root IAA oxidase activity. This response, leads to, or at least partially leads to an increase in root IAA levels, which in turn help induce increased root FCR activity.Key Words: Fe deficiency, ferric chelate reductase, phenolics, Trifolium pretense     

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 deficiency enhances the levels of ascorbate,glutathione, and related enzymes in sugar beet roots

Summary.  The effects of Fe deficiency stress on the levels of ascorbate and glutathione, and on the activities of the enzymes ferric chelate reductase, glutathione reductase (EC 1.6.4.2), ascorbate free-radical reductase (EC 1.6.5.4) and ascorbate peroxidase (EC 1.11.1.11), have been investigated in sugar beet (Beta vulgaris L.) roots. Plasma membrane vesicles and cytosolic fractions were isolated from the roots of the plants grown in nutrient solutions in the absence or presence of Fe for two weeks. Plants responded to Fe deficiency not only with a 20-fold increase in root ferric chelate reductase activity, but also with moderately increased levels of the general reductants ascorbate (2-fold) and glutathione (1.6-fold). The enzymes of the ascorbate-glutathione cycle in roots were also affected by Fe deficiency. Glutathione reductase activity was enhanced 1.4-fold with Fe deficiency, associated to an increased ratio of reduced to oxidized glutathione, from 3.1 to 5.2. The plasma membrane fraction from iron-deficient roots showed 1.7-fold higher ascorbate free-radical reductase activity, whereas in the cytosolic fraction the enzyme activity was not affected by Fe deficiency. The activity of the cytosolic hemoprotein ascorbate peroxidase decreased approximately by 50% with Fe deprivation. These results show that sugar beet responds to Fe deficiency with metabolic changes affecting components of the ascorbate-glutathione cycle in root cells. This suggests that the ascorbate-glutathione cycle would play certain roles in the general Fe deficiency stress responses in strategy I plants. Received November 19, 2001; accepted September 30, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, CSIC, Apartado 202, 50080 Zaragoza, Spain.     

Kinetic properties of a micronutrient transporter from<Emphasis Type="Italic"> Pisum sativum</Emphasis> indicate a primary function in Fe uptake from the soil

Fe uptake in dicotyledonous plants is mediated by a root plasma membrane-bound ferric reductase that reduces extracellular Fe(III)-chelates, releasing Fe²⁺ ions, which are then absorbed via a metal ion transporter. We previously showed that Fe deficiency induces an increased capacity to absorb Fe and other micronutrient and heavy metals such as Zn²⁺ and Cd²⁺ into pea (Pisum sativum L.) roots [Cohen et al. (1998) Plant Physiol 116:1063–1072). To investigate the molecular basis for this phenomenon, an Fe-regulated transporter that is a homologue of the Arabidopsis IRT1 micronutrient transporter was isolated from pea seedlings. This cDNA clone, designated RIT1 for root iron transporter, encodes a 348 amino acid polypeptide with eight putative membrane-spanning domains that is induced under Fe deficiency and can functionally complement yeast mutants defective in high- and low-affinity Fe transport. Chelate buffer techniques were used to control Fe²⁺ in the uptake solution at nanomolar activities representative of those found in the rhizosphere, and radiotracer methodologies were employed to show that RIT1 is a very high-affinity ⁵⁹Fe²⁺ uptake system (K _m =54–93 nM). Additionally, radiotracer (⁶⁵Zn, ¹⁰⁹Cd) flux techniques were used to show that RIT can also mediate a lower affinity Zn and Cd influx (K _m of 4 and 100 M, for Zn²⁺ and Cd²⁺, respectively). These findings suggest that, in typical agricultural soils, RIT1 functions primarily as a high-affinity Fe²⁺ transporter that mediates root Fe acquisition. This is consistent with recent findings with Arabidopsis IRT1 knockout mutants that strongly suggest that this transporter plays a key role in root Fe uptake and nutrition. However, the ability of RIT1 to facilitate Zn and Cd uptake when these metals are present at elevated concentrations suggests that RIT1 may be one pathway for the entry of toxic metals into the food chain. Furthermore, the finding that plant Fe deficiency status may promote heavy metal uptake via increased expression of this transporter could have implications both for human nutrition and also for phytoremediation, the use of terrestrial plants to sequester toxic metals from contaminated soil.     

Arabidopsis cpFtsY mutants exhibit pleiotropic defects including an inability to increase iron deficiency-inducible root Fe(III) chelate reductase activity

All plants, except for the grasses, must reduce Fe(III) to Fe(II) in order to acquire iron. In Arabidopsis, the enzyme responsible for this reductase activity in the roots is encoded by FRO2. Two Arabidopsis mutants, frd4-1 and frd4-2, were isolated in a screen for plants that do not induce Fe(III) chelate reductase activity in their roots in response to iron deficiency. frd4 mutant plants are chlorotic and grow more slowly than wild-type Col-0 plants. Additionally, frd4 chloroplasts are smaller in size and possess dramatically fewer thylakoid membranes and grana stacks when compared with wild-type chloroplasts. frd4 mutant plants express both FRO2 and IRT1 mRNA normally in their roots under iron deficiency, arguing against any defects in systemic iron-deficiency signaling. Further, transgenic frd4 plants accumulate FRO2-dHA fusion protein under iron-deficient conditions, suggesting that the frd4 mutation acts post-translationally in reducing Fe(III) chelate reductase activity. FRO2-dHA appears to localize to the plasma membrane of root epidermal cells in both Col-0 and frd4-1 transgenic plants when grown under iron-deficient conditions. Map-based cloning revealed that the frd4 mutations reside in cpFtsY, which encodes a component of one of the pathways responsible for the insertion of proteins into the thylakoid membranes of the chloroplast. The presence of cpFtsY mRNA and protein in the roots of wild-type plants suggests additional roles for this protein, in addition to its known function in targeting proteins to the thylakoid membrane in chloroplasts.     

Ethylene involvement in the over-expression of Fe(III)-chelate reductase by roots of E107 pea [Pisum sativum L. (brz, brz)] and chloronerva tomato (Lycopersicon esculentum L.) mutant genotypes

Recently, ethylene was reported to be involved in the regulation of Fe(III)-chelate reducing capacity by cucumber (Cucuinis sativus L.) roots. Here, we studied the effect of two ethylene inhibitors, aminooxyacetic acid (AOA) and cobalt, on the Fe(III) reducing capacity in roots of mutant genotypes [E107 pea [Pisum sativum L. (brz, brz)] and chloronerva tomato (Lycopersicon esculentum L.) that exhibit high rates of Fe(III)-chelate reduction and excessive iron accumulation. The ethylene inhibitors, AOA and cobalt, markedly inhibited Fe(III)-chelate reducing capacity in roots of both genotypes. Over-expression of root Fe(III) reductase activity by both mutants appears to be related to ethylene. Possibly, both mutants are genetically defective in their ability to regulate root ethylene production. The large inhibitory effect of both ethylene inhibitors on Fe(III)-chelate reducing capacity in roots of the mutant tomato genotype, chloronerva, disputes the contention that the nicotianamine-Fe(II) complex is the repressior of the gene responsible for Fe(III)-chelate reductase activity, as previously suggested by others. However, since nicotianamine shares the same biosynthetic precursor as ethylene, i.e. S-adenosyl methionine, nicotianamine may affect Fe(III)-chelate reductase activity in dicot and non-grass monocot roots by influencing ethylene biosynthesis.     

The CorA Mg2+ transporter does not transport Fe2+

corA encodes the constitutively expressed primary Mg2+ uptake system of most eubacteria and many archaea. Recently, a mutation in corA was reported to make Salmonella enterica serovar Typhimurium markedly resistant to Fe2+-mediated toxicity. Mechanistically, this was hypothesized to be from an ability of CorA to mediate the influx of Fe2+. Consequently, we directly examined Fe2+ transport and toxicity in wild-type versus corA cells. As determined by direct transport assay, CorA cannot transport Fe2+ and Fe2+ does not potently inhibit CorA transport of 63Ni2+. Mg2+ can, relatively weakly, inhibit Fe2+ uptake, but inhibition is not dependent on the presence of a functional corA allele. Although excess Fe2+ was slightly toxic to S. enterica serovar Typhimurium, we were unable to elicit a significant differential sensitivity in a wild-type versus a corA strain. We conclude that CorA does not transport Fe2+ and that the relationship, if any, between iron toxicity and corA is indirect.     

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⁺.     

Abscisic acid alleviates iron deficiency by promoting root iron reutilization and transport from root to shoot in Arabidopsis

Abscisic acid (ABA) has been demonstrated to be involved in iron (Fe) homeostasis, but the underlying mechanism is largely unknown. Here, we found that Fe deficiency induced ABA accumulation rapidly (within 6 h) in the roots of Arabidopsis. Exogenous ABA at 0.5 μM decreased the amount of root apoplastic Fe bound to pectin and hemicellulose, and increased the shoot Fe content significantly, thus alleviating Fe deficiency‐induced chlorosis. Exogenous ABA promoted the secretion of phenolics to release apoplastic Fe and up‐regulated the expression of AtNRAMP3 to enhance reutilization of Fe stored in the vacuoles, leading to a higher level of soluble Fe and lower ferric–chelate reductase (FCR) activity in roots. Treatment with ABA also led to increased Fe concentrations in the xylem sap, partially because of the up‐regulation of AtFRD3, AtYSL2 and AtNAS1, genes related to long‐distance transport of Fe. Exogenous ABA could not alleviate the chlorosis of abi5 mutant resulting from the significantly low expression of AtYSL2 and low transport of Fe from root to shoot. Taken together, our data support the conclusion that ABA is involved in the reutilization and transport of Fe from root to shoot under Fe deficiency conditions in Arabidopsis.     

Role of hormones in the induction of iron deficiency responses in Arabidopsis roots

In "strategy I" plants, several alterations in root physiology and morphology are induced by Fe deficiency, although the mechanisms by which low Fe levels are translated into reactions aimed at alleviating Fe shortage are largely unknown. To prove whether changes in hormone concentration or sensitivity are involved in the adaptation to suboptimal Fe availability, we tested 45 mutants of Arabidopsis defective in hormone metabolism and/or root hair formation for their ability to increase Fe(III) chelate reductase activity and to initiate the formation and enlargement of root hairs. Activity staining for ferric chelate reductase revealed that all mutants were responsive to Fe deficiency, suggesting that hormones are not necessary for the induction. Treatment of wild-type plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid caused the development of root hairs in locations normally occupied by non-hair cells, but did not stimulate ferric reductase activity. Ectopic root hairs were also formed in -Fe roots, suggesting a role for ethylene in the morphological responses to Fe deficiency. Ultrastructural analysis of rhizodermal cells indicated that neither Fe deficiency nor 1-aminocyclopropane-1-carboxylic acid treatment caused transfer-cell-like alterations in Arabidopsis roots. Our data indicate that the morphological and physiological components of the Fe stress syndrome are regulated separately.