Characterization and in vitro selection for iron efficiency inPyrus andCydonia

Summary Responses to low Fe were characterized in tissue cultures ofPyrus amygdaliformis andCydonia oblonga (quince), two species used as rootstocks for pear. Cultured shoots and plantlets ofP. amygdaliformis had a higher chlorophyll concentration and Fe²⁺/total Fe ratio than those ofC. oblonga when grown under low Fe conditions. This tolerance to low Fe was correlated with high Fe³⁺-reducing ability and medium acidification. The adaptive responses were manifested in roots of plantlets, shoot bases, root cultures, and cell suspension cultures. Shoots were regenerated from leaves of quince and subjected to Fe-deficient conditions. Two somaclonal variants (IE-1 and IE-2) were recovered; each displayed higher ability to reduce Fe³⁺ and acidify the medium. These variants may be useful as rootstocks for regions with calcareous soils, which limit Fe availability.     

Pear plantlets cultured ‘in vitro’ under lime-induced chlorosis display a better adaptive strategy than quince plantlets

In many fruit trees species lime-induced chlorosis causes serious economic damage. In the last few years, the in vitro culture technique has been applied to test rootstocks for susceptibility to iron chlorosis and to study biochemical and molecular aspects of the syndrome. In this study in vitro shoot cultivation of quince rootstocks MA, BA29 and pear cv. ‘Conference’ was used to unravel the relationship between iron deficiency, presence of bicarbonate, and growth development and chloroplast pigment behaviour. Stresses were obtained by using MS medium supplemented with various concentrations of FeNaEDTA and/or KHCO₃. Substrate pH, shoot growth and development parameters, total leaf iron and photosynthetic pigment contents of plantlets were independently determined after 24 days of culture. The medium was acidified by the species under all stress conditions. Iron deficiency and bicarbonate condition led to different growth patterns and modular development among the genotypes. This reflects a different sensitivity and plastic adaptation to the elements of this stress. Iron concentration in leaves and the shoot apex of plantlets decreased in all genotypes. However, chloroplast pigments only decreased in quince plantlets under iron deficiency and bicarbonate condition while they did not change in pear plantlets. Our results demonstrate firstly that a plantlet without a root system, which is cultivated in vitro, is able to sense iron deficiency and bicarbonate enriched conditions and, consequently, activates biochemical and physiological responses. Secondly, acidification appears to be related to iron concentration in tissues of quince rootstocks and pear cultivar. Finally, chloroplast biochemical elements are strongly and differently regulated under both stress conditions and between genotypes.     

Iron toxicity and stress-induced ethylene production in rice leaves

The relationship among iron toxicity, bronzing symptom, and stress-induced ethylene production (SEP) was investigated in detached rice (Oryza sativa L.) leaves during the vegetative-ripening stage and in whole plants during the vegetative stage. When Fe²⁺ (200 mg L^-1) was applied to the detached leaf through a transpiration stream, SEP was higher in the first leaf than in the second and third leaves from the top and maximal around the panicle primordia initiation stage. The genotype difference in SEP was more pronounced in the second and third leaves than in the first leaf. Bronzing intensity increased as SEP increased; iron concentration increase during treatment in the tissue did not correlate with bronzing intensity or with SEP among the 16 genotypes tested. When the roots of an intact plant were exposed to 300 mg L^-1 of Fe²⁺ in culture solution little stress-induced ethylene was produced. By partially or totally derooting the plant, however, stress-induced ethylene was evoked, indicating that roots reduced the Fe²⁺ uptake so that little stress ethylene is produced in the intact plant. Leaf tissue tolerance for Fe²⁺ may contribute to genotype differences in iron toxicity tolerance of rice plants when roots are injured during transplanting or exposed to toxic substances in the soil.     

Effects of free and chelated iron on in vitro androgenesis in barley and wheat

A suitable form of iron supplement in the induction medium was found to be important for further development of induced pollen embryos in barley and wheat cultivars (genotypes), especially those providing few green plants viain vitro androgenesis. Genotypes able to regenerate many green plants were less susceptible to the lack of iron in induction medium. Although Fe-EDTA was found to be a suitable form of iron in the induction medium, androgenesis was also induced on media containing non-chelated iron (Fe²⁺ and Fe³⁺ ions). EDTA alone without iron inhibited the androgenic response even in the wheat cv. Florida, a model cultivar for androgenesis in wheat. In all barley cultivars under study including cv. Igri, a model cultivar for androgenesis in barley, EDTA alone caused an almost total suppression of androgenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morphological and physiological responses of root tip cells to Fe<Superscript>2+</Superscript> toxicity in rice

Two genotypes of rice (Oryza sativa L.), Azucena (iron tolerant) and IR64 (iron sensitive), were used to investigate the numbers and survival rates of root border cells (namely, in situ border cells) in plants that were exposed to excess iron (Fe²⁺). Additionally, we examined the changes in the root tip cell morphology and activities of protective enzymes in response to Fe²⁺ toxicity. The results showed that Fe²⁺ toxicity hindered the development of root border cells (RBCs) and that higher Fe²⁺ concentrations caused root cap cell walls to thicken. In the iron-sensitive rice variety, these changes lowered RBC survival rate and lead to programmed cell death. Low concentrations of Fe²⁺ were shown to facilitate the development of RBCs in the iron-tolerant rice variety and that the activities of the protective enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were elevated in the iron-tolerant variety, thus suggesting that rice root tips could defend against Fe²⁺ toxicity by producing RBCs, root cap cells, and protective enzymes.     

钒钛磁铁尾矿土壤中水黄皮根瘤菌铁耐受性与钝化能力研究

铁是好氧微生物生长所必需的元素,而铁污染土壤环境中的根瘤菌是否对高浓度铁具有耐受性和钝化能力尚不清楚。以攀枝花钒钛磁铁尾矿土壤作为基质进行水黄皮共生根瘤菌捕获实验,获得水黄皮共生根瘤并从中分离纯化出根瘤菌39株。通过Fe~(2+)/Fe~(3+)耐受性和钝化能力测试筛选出耐受性和钝化能力均强的优势菌株PZHS20、PZHS90、PZHS87,其对Fe~(2+)的最大耐受质量浓度为1 600 mg/L,其中PZHS20在200 mg/L Fe~(2+)溶液中钝化效率最大,为73.54%;PZHS90对Fe~(3+)的最大耐受质量浓度为1 600 mg/L,而PZHS20和PZHS87对Fe~(3+)的最大耐受质量浓度为1 800 mg/L,其在200 mg/L Fe~(3+)溶液中钝化效率分别为84.25%和81.95%。16S rRNA基因系统进化分析将PZHS20鉴定为苍白杆菌(Ochrobactrum),将PZHS90和PZHS87鉴定为慢生根瘤菌(Bradyrhizobium)。研究结果表明,钒钛磁铁尾矿土壤中的水黄皮根瘤菌具有不同程度的Fe~(2+)/Fe~(3+)耐受性和钝化能力,筛选出的优势菌株为进一步利用水黄皮-根瘤菌联合修复高浓度铁污染土壤提供可利用的菌株资源。     

Evidence for chemical changes on the root surface of tall fescue in response to infection with the fungal endophyte Neotyphodium coenophialum

Endophyte-infected (E+) tall fescue (Festuca arundinacea Schreb.) plants grown in phosphorus (P) deficient soils accumulate more P in roots and shoots than noninfected isolines. In a growth chamber experiment, four tall fescue genotypes DN2, DN4, DN7, and DN11, infected with their naturally occurring strains of Neotyphodium coenophialum (Morgan-Jones & Gams) Glenn, Bacon & Hanlin, and their noninfected isolines (E-), were cultivated in nutrient solution at two P levels: 31 ppm (P+) and 0 ppm (P-) for 4 wk. The Fe³⁺ reducing activity of extracellular reductants and intact root tissues, and total phenolic concentration in roots and shoots were measured. Endophyte infection significantly increased Fe³⁺ reducing activity rate of extracellular reductants (9.6 × 10^-3 mol Fe³⁺ h-1 g-1 root FW) when compared to E- plants (3.9 × 10^-3) and Fe³⁺ reduction rate of intact root tissues (6.16 and 4.48 mol Fe³⁺ h-1 g-1 root FW, respectively for E+ and E- plants). In response to P deficiency, Fe³⁺ reduction rate of intact root tissues increased in E+ plants by 375% when compared to E- plants, whereas no significant differences were observed when P was provided. Total phenolic concentration was 20% greater in shoots of E+ plants than in E- plants. In response to P deficiency, total phenolic concentration significantly increased in roots of E+ plants by 7%, and decreased in roots of E- plants by 10%. The most active Fe³⁺ reducing zones were located along branching of secondary and tertiary roots. The Fe³⁺ reducing activity on the root surface and total phenolic concentration in roots and shoots increased dramatically in response to endophyte infection, especially under P limiting conditions.Visiting Scientist sponsored by the Fulbright Program No. 21133     

Metabolic response in roots of Prunus rootstocks submitted to iron chlorosis

Iron deficiency induces several responses to iron shortage in plants. Metabolic changes occur to sustain the increased iron uptake capacity of Fe-deficient plants. We evaluated the metabolic changes of three Prunus rootstocks submitted to iron chlorosis and their different responses for tolerance using measurements of metabolites and enzymatic activities. The more tolerant rootstocks Adesoto (Prunus insititia) and GF 677 (Prunus amygdalus × Prunus persica), and the more sensitive Barrier (P. persica × Prunus davidiana) were grown hydroponically in iron-sufficient and -deficient conditions over two weeks. Sugar, organic and amino acid concentrations of root tips were determined after two weeks of iron shortage by proton nuclear magnetic resonance spectroscopy of extracts. Complementary analyses of organic acids were performed by liquid chromatography coupled to mass spectrometry. The major soluble sugars found were glucose and sucrose. The major organic acids were malic and citric acids, and the major amino acid was asparagine. Iron deficiency increased root sucrose, total organic and amino acid concentrations and phosphoenolpyruvate carboxylase activity. After two weeks of iron deficiency, the malic, citric and succinic acid concentrations increased in the three rootstocks, although no significant differences were found among genotypes with different tolerance to iron chlorosis. The tolerant rootstock Adesoto showed higher total organic and amino acid concentrations. In contrast, the susceptible rootstock Barrier showed lower total amino acid concentration and phosphoenolpyruvate carboxylase activity values. These results suggest that the induction of this enzyme activity under iron deficiency, as previously shown in herbaceous plants, indicates the tolerance level of rootstocks to iron chlorosis. The analysis of other metabolic parameters, such as organic and amino acid concentrations, provides complementary information for selection of genotypes tolerant to iron chlorosis.     

Investigations on iron uptake and reduction by excised roots of different grapevine rootstocks and a V. vinifera cultivar

Roots from cuttings of grapevine rootstocks V. Berlandieri × V. rupestris 140 Ru, V. Berlandieri × V. riparia SO4, V. riparia × V. rupestris 101–14 and a V. vinifera cultivar (Pinot blanc), with a decreasing degree of chlorosis resistance, were excised and tested to quantify physiological and morphological parameters, as follows: ⁵⁹Fe uptake from an FeEDTA solution; reducing capacity; root diameter and root hair occurrence.The most significant findings are that: a) ⁵⁹Fe uptake and FeEDTA reduction decreased from the most chlorosis resistant rootstock (140 Ru) to the most susceptible one (101–14) and to Pinot blanc; b) ⁵⁹Fe uptake and reducing capacity were closely related; c) iron uptake and reduction were closely related to the root diameter and root hair occurrence, within the rootstocks.     

The calcium nutrition of bambara groundnut (Vigna subterranea (L.) Verdc.)

Iron chlorosis induced by Fe-deficiency is a widespread nutritional disorder in many woody plants and in particular in grapevine. This phenomenon results from different environmental, nutritional and varietal factors. Strategy I plants respond to Fe-deficiency by inducing physiological and biochemical modifications in order to increase Fe uptake. Among these, acidification of the rhizosphere, membrane redox activities and synthesis of organic acids are greatly enhanced during Fe-deficiency. Grapevine is a strategy I plant but the knowledge on the physiological and biochemical responses to this iron stress deficiency in this plant is still very poor. In this work four different genotypes of grapevine were assayed for these parameters. It was found that there is a good correlation between genotypes which are known to be chlorosis-resistant and increase in both rhizosphere acidification and Fe^III reductase activity. In particular, when grown in the absence of iron, Vitis berlandieri and Vitis vinifera cv Cabernet sauvignon and cv Pinot blanc show a higher capacity to acidify the culture medium (pH was decreased by 2 units), a higher concentration of organic acids, a higher resting transmembrane electrical potential and a greater capacity to reduce Fe^III-chelates. On the contrary, Vitis riparia, well known for its susceptibility to iron chlorosis, fails to decrease the pH of the medium and shows a lower concentration in organic acids, lower capacity to reduce Fe^III and no difference in the resting transmembrane electrical potential. H Marschner Section editor     

Characterization of the tolerance to iron chlorosis in different peach rootstocks grown in nutrient solution

Iron chlorosis is an important problem in peach trees, but differences exist between peach rootstocks in their tolerance to Fe chlorosis in calcareous soils. The purpose of this investigation was to characterize the tolerance of different rootstocks to Fe chlorosis induced by bicarbonate in nutrient solution. The rootstocks studied included peach (Nemaguard), plums (Brompton, San Julian A and Puebla de Soto 101) and almond × peach hybrids (Adafuel and GF677). Young plants obtained from rooted cuttings or from in vitro culture techniques were grown individually, under controlled conditions, in flasks with 700 mL of aerated nutrient solution low in iron and with or without 10 mM bicarbonate or 10 mM phosphate. Susceptiblity to bicarbonate-induced chlorosis was inversely correlated with both the Fe content in young leaves and the reducing capacity of roots, but not with the phosphorus content in young leaves. The plum Puebla de Soto 101 and the hybrid GF677 showed the lowest degree of chlorosis and the highest reducing capacity. Phosphate did not induce chlorosis.     

Differences in responses to iron deficiency among various cultivars of mungbean (Vigna radiata (L.) Wilczek)

Ten mungbean cultivars were evaluated for their resistance to iron deficiency in view of chlorosis symptoms, plant growth and seed yield under field conditions on a calcareous soil in Thailand. The KPS2 cultivar was highly susceptible; the KPS1, PSU1 and Pag-asa 1 cultivars were somewhat susceptible; the VC1163B cultivar was moderately tolerant; the CN36, CN60, UT1 and CNM-I cultivars were tolerant; and the CNM8509B cultivar was very tolerant to iron deficiency. Foliar application of a solution of 5 g L^-1 ferrous sulphate was effective in correcting chlorosis that was induced by iron deficiency, and it enhanced both the growth and the yield of susceptible cultivars. Compared with the susceptible cultivar KPS2, the tolerant cultivar UT1 had a greater ability to lower the pH of the nutrient solution in response to iron deficiency. The root-associated Fe³⁺-reduction activity of UT1 that had been grown in -Fe medium was similar to that of the plants grown in +Fe medium when the acidification of the medium occurred. Acidification of the medium in response to iron deficiency might contribute to the efficient solubilization of iron from calcareous soils, and it related more closely to the resistance to iron deficiency than Fe³⁺ reduction by roots in mungbean cultivars.     

Evaluation of early generations for iron chlorosis in relation to productivity in groundnut (Arachis hypogaea L.)

Five popular but iron-inefficient cultivars were crossed with three efficient genotypes and both parents and F₁s were evaluated for iron-efficiency in potted calcareous and noncalcareous soil. The iron-efficient genotypes were dark green or green in both noncalcareous and calcareous soils whereas inefficient types were light green to yellow in calcareous soil. The chlorophyll and active iron (Fe²⁺) concentration of leaves was less in iron-efficient genotypes compared to efficient types in calcareous soil and reduction of both the parameters from noncalcareous to calcareous soil was considerably high in iron-inefficient lines. There was significant correlation between visual scores, chlorophyll and active iron content. There were no differences among F₁s for iron chlorosis and they were all iron-inefficient. The frequency of iron-inefficient plants was higher than the efficient plants in all F₂ populations. But most of the productive plants came from iron-efficient segregants indicating strong association between iron-efficiency and productivity. Based on the results selection for iron-efficiency in early generations and extensive evaluation for productivity in advanced generations is suggested for developing varieties for cultivation in calcareous soils.     

Iron-Stress Response in Tomato (Lycopersicon esculentum) 1. Sites of Fe Reduction, Absorption and Transport

T3238fer (Fe-inefficient) and T3238FER (Fe-efficient) tomato plants differ in their ability to utilize Fe and therefore can be used as test genotypes to locate sites of Fe uptake or to characterize changes that occur in roots in response to Fe stress (Fe deficiency). T3238fer does not respond to Fe stress. Release of hydrogen ions and reduction of Fe³⁺ to Fe²⁺ are two primary responses of T3238FER roots to Fe stress. Fe reduction sites were predominately in the young lateral roots, and between the regions of root elongation and maturation of the primary root. The use of BDPS (bathophenanthrolinedisulfonate) to trap Fe²⁺ did not affect the release of H⁺ ions or reduction by T3238FER roots. BPDS did not decrease Fe uptake until it exceeded the Fe concentration in the nutrient solution. A sevenfold increase in BPDS caused a threefold decrease in Fe taken up by the plant. Fe³⁺ is reduced to Fe²⁺ at root sites accessible to BPDS. Adding Zn decreased the response to Fe stress. Iron stress initiates the development of lateral roots, and we propose that most Fe enters the plant through these roots. The iron moves through protoxylem into the metaxylem of the primary root and then to the top of the plant as Fe citrate. Root environmental factors that are competitive or inhibit Fe-stress response, or genotypes that fail to respond to Fe stress, contribute to the development of Fe deficiency in plants.     

不同浓度Fe2+与Zn2+对羊草幼苗生长和生理特性的影响

以羊草幼苗为研究对象,通过调整全营养培养基(CK,0.05 mmol/L Fe²⁺、0.015 mmol/L Zn²⁺)中铁或者锌含量设置0、10倍、20倍Fe²⁺(Zn²⁺)浓度处理Fe₀(Zn₀)、Fe₁₀(Zn₁₀)、Fe₂₀(Zn₂₀),以及在高铁培养基中单独添加0.15 mmol/L Zn²⁺或同时添加10 mmol/L Ca²⁺、5 mmol/L Mg²⁺、20 mmol/L K⁺处理,测定培养6 d后幼苗生长指标和矿质元素含量、以及高铁(Fe₂₀)处理下幼苗根中抗氧化指标和相关基因表达量,探究不同浓度Fe²⁺、Zn²⁺对羊草幼苗生长、矿质元素吸收积累及抗氧化指标、基因表达的影响。结果表明：(1)缺锌(Zn₀)显著抑制羊草幼苗鲜重的增加和Zn元素的积累,但促进Fe、Mg元素的积累;高浓度锌(Zn₁₀、Zn₂₀)显著促进幼苗叶片生长和Zn元素的积累;缺铁(Fe₀)显著抑制幼苗的根长、鲜重和Fe元素的积累,促进Mg、Zn元素的积累;高浓度铁(Fe₁₀、Fe₂₀)显著抑制羊草幼苗根叶生长、根毛发育和Ca、Zn、Mg、K元素的积累。(2)增加Zn²⁺和Ca²⁺、Mg²⁺、K⁺浓度无法恢复高铁胁迫对幼苗生长的抑制作用。(3)高浓度铁(Fe₂₀)处理羊草幼苗48 h后,根部过氧化物酶、超氧化物歧化酶、过氧化氢酶、抗坏血酸过氧化物酶、谷胱甘肽还原酶活性和丙二醛、抗坏血酸、还原型谷胱甘肽含量显著升高;烟酰胺合成酶基因、过氧化物酶基因表达量显著下调,植物类萌发素蛋白基因表达量显著上调。研究发现,羊草幼苗生长发育和矿质元素积累对环境中Zn²⁺浓度变化不敏感,却受到环境中高浓度Fe²⁺的显著抑制,并造成严重的氧化胁迫伤害,这种伤害无法在添加Zn²⁺或同时添加Ca²⁺、Mg²⁺、K⁺的条件下恢复。     

Conventional and transgenic strategies to enhance the acid soil tolerance of barley

The aluminum (Al³⁺) tolerance of barley cultivars predominately from Brazil was compared to that of cultivars from other countries, wild barley accessions, and a transgenic line (L5) over-expressing TaALMT1, the major Al³⁺ tolerance gene from wheat. After screening conventional germplasm for Al³⁺ tolerance in hydroponics, 18 genotypes were further characterized in a short-term soil experiment. Among the Brazilian cultivars, Antarctica 01 and BRS Mariana showed the greatest relative root length (RRL) in acid soil. However, these cultivars were significantly less tolerant than the foreign cultivars Dayton (USA) and Murasakimochi (Japan) and the transgenic line L5 which out-performed all conventional genotypes. In long-term growth trials, the transgenic line produced the greatest relative root and relative shoot dry weight. Relative grain yield was greatest in the transgenic line and Dayton. All genotypes were also scored for two genetic markers linked to HvAACT1, the major Al³⁺ tolerance gene in barley. One marker detects a 1-kb insertion in the promoter that increases gene expression and leads to increased Al³⁺-activated citrate efflux from root apices. The other marker detects a 21-bp indel downstream of the coding region. The 1-kb insertion was only detected in Dayton and Murasakimochi that were the best performing cultivars among the non-transgenic germplasm. Interestingly, the Brazilian cultivars with an intermediate level of tolerance, Antarctica 01 and BRS Mariana, lacked the 1-kb insertion but had enhanced HvAACT1 expression compared to an Al³⁺-sensitive cultivar. No clear correlation was observed between Al³⁺ tolerance and the 21-bp indel marker in the short-term soil trials. We conclude that improved Al³⁺ tolerance in barley could be achieved by combining the best allele of HvAACT1 along with TaALMT1 as a transgene.     

pH Changes Associated with Iron-Stress Response

When Fe-inefficient T3238fer and Fe-efficient T3238FER tomatoes were supplied iron, and nitrogen as nitrate, they increased the pH of the nutrient culture. When they were supplied nitrogen as ammonium, they decreased the pH. When Fe supply was limited, Fe-stress response developed in T3238FER that opposed the usual nitrate response and decreased, rather than increased, the pH. A “reductant” which reduced Fe³⁺ to Fe²⁺ was released from the roots of these plants and lowered the pH; and there was a tremendous increase in the uptake of Fe. T3238fer did not produce “reductant” in response to Fe-stress; the pH increased, and the plants developed Fe-deficiency when nitrogen was supplied as nitrate. Nitrogen nutrition and iron-stress response are important factors associated with iron chlorosis in plants. Release of hydrogen ions from roots of Fe-stressed plants is caused by more than response to imbalanced uptake of cations and anions.     

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.     

Plasma membrane-bound NADH: Fe3+-EDTA reductase and iron deficiency in tomato (Lycopersicon esculentum). Is there a Turbo reductase?

The properties of NADH-dependent Fe³⁺-EDTA reductase in plasma membranes (PM) from roots of iron-deficient and -sufficient tomato plants [Lycopersicon esculentum L. (Mill.) cv. Abunda] were examined. Iron deficiency resulted in a 3-fold increase of in vivo root iron-chelate reductase activity with a K_m (Fe³⁺-EDTA) of 230 μM. In purified root PM, average specific activities of ferric chelate reductase of 410 and 254 nmol Fe (mg protein)^?1 min^?1 were obtained for iron-deficient and -sufficient plants, respectively. In both cases, the PM-bound activity showed a pH optimum at pH 6.8. Activity depended on NADH and not on NADPH and on the presence of detergent. The activity was inhibited 40-50% by superoxide dismutase (EC 1.15.1.1) and ca 30% by oxygen. Kinetic analysis of the membrane-bound enzyme revealed a K_m (Fe³⁺-EDTA) of ca 200 μM for both iron-stressed and -sufficient plants. For NADH, K_m values around 230 μM were obtained. The ferric chelate reductase could be solubilised from salt-washed PM with Triton X-100 at a protein:detergent ratio of 1:2.8 (w/w). The Triton-soluble fraction revealed one enzyme-stained band in native polyacrylamide electrophoresis. Although the membranes showed no nitrate reductase (NR; EC 1.6.6.1) activity, anti-spinach NR immunoglobulin G (IgG) recognized a 54 kDa band both in the PM and the Triton-soluble fraction, but not in the enzymatically active material obtained from the native gel. No evidence could be found for the synthesis of a new, biochemically distinct PM-bound ferric chelate reductase under iron deficiency, which might be identified as the so-called Turbo reductase. It is concluded that iron deficiency in tomato induces increased expression of a ferric chelate reductase in root PM, which is already present in iron-sufficient plants and probably also in plants, which do not contain the Turbo reductase, like the grasses. The iron reductase is not identical with the recently reported PM-associated nitrate reductase.     

Dynamic equilibria in iron uptake and release by ferritin

The function of ferritins is to store and release ferrous iron. During oxidative iron uptake, ferritin tends to lower Fe²⁺ concentration, thus competing with Fenton reactions and limiting hydroxy radical generation. When ferritin functions as a releasing iron agent, the oxidative damage is stimulated. The antioxidant versus pro-oxidant functions of ferritin are studied here in the presence of Fe²⁺, oxygen and reducing agents. The Fe²⁺-dependent radical damage is measured using supercoiled DNA as a target molecule. The relaxation of supercoiled DNA is quantitatively correlated to the concentration of exogenous Fe²⁺, providing an indirect assay for free Fe²⁺. After addition of ferrous iron to ferritin, Fe²⁺ is actively taken up and asymptotically reaches a stable concentration of 1–5 m. Comparable equilibrium concentrations are found with plant or horse spleen ferritins, or their apoferritins. After addition of ascorbate, iron release is observed using ferrozine as an iron scavenger. Rates of iron release are dependent on ascorbate concentration. They are about 10 times larger with pea ferritin than with horse ferritin. In the absence of ferrozine, the reaction of ascorbate with ferritins produces a wave of radical damage; its amplitude increases with increased ascorbate concentrations with plant ferritin; the damage is weaker with horse ferritin and less dependent on ascorbate concentrations.