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.     

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.     

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.     

Iron limitation results in induction of ferricyanide reductase and ferric chelate reductase activities in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii Dangeard CW-15 exhibited very low rates of plasma-membrane Fe(III) reductase activity when grown under Fe-sufficient conditions. After switching the medium to an Fe-free formulation, both ferricyanide reductase and ferric chelate reductase activities rapidly increased, reaching a maximum after 3 d under iron-free conditions. Both of the Fe(III) reductase activities increased in parallel over time, they exhibited similar K _m values (approximately 10 μM) with respect to Fe(III), displayed the same pH profile of activity, and both exhibited the same degree of light stimulation which could be inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). Furthermore, ferricyanide competitively inhibited ferric chelate reduction by iron-limited cells. These results indicate that both Fe(III) reductase activities were mediated by the same iron-limitation-induced plasma-membrane reductase. No evidence was found for the presence of Fe(III)-reducing substances in the culture medium, or for the involvement of active oxygen species in the process of Fe(III) reduction. Chlamydomonas reinhardtii appears to respond to iron limitation in a manner similar to Strategy I higher plants. Received: 24 June 1997 / Accepted: 2 August 1997     

Responses of Arabidopsis thaliana to bicarbonate-induced iron deficiency

Morpho-physiological and biochemical responses of Arabidopsis thaliana (accession N1438) to bicarbonate-induced iron deficiency were investigated. Plants were grown in cabinet under controlled conditions, in a nutrient solution containing 5 μM Fe, added or not with 10 mM NaHCO₃. After 30 days, bicarbonate-treated plants displayed significantly lower biomass, leaf number and leaf surface area as compared to control plants, and slight yellowing of their younger leaves was observed. Potassium (K⁺) content was not modified by bicarbonate treatment in roots, whereas it was significantly diminished in shoots. Their content in ferrous iron (Fe²⁺) and in leaf total chlorophylls was noticeably lower than in control plants. Root Fe(III)-chelate reductase and phosphoenolpyruvate carboxylase (PEPC) activities were significantly enhanced, but leaf ribulose 1.5-bisphosphate carboxylase (Rubisco) activity was decreased.     

Iron stress-induced changes in root epidermal cell fate are regulated independently from physiological responses to low iron availability

Iron-overaccumulating mutants were investigated with respect to changes in epidermal cell patterning and root reductase activity in response to iron starvation. In all mutants under investigation, ferric chelate reductase activity was up-regulated both in the presence and absence of iron in the growth medium. The induction of transfer cells in the rhizodermis appeared to be iron regulated in the pea (Pisum sativum L. cv Dippes Gelbe Viktoria and cv Sparkle) mutants bronze and degenerated leaflets, but not in roots of the tomato (Lycopersicon esculentum Mill. cv Bonner Beste) mutant chloronerva, suggesting that in chloronerva iron cannot be recognized by putative sensor proteins. Experiments with split-root plants supports the hypothesis that Fe(III) chelate reductase is regulated by a shoot-borne signal molecule, communicating the iron status of the shoot to the roots. In contrast, the formation of transfer cells was dependent on the local concentration of iron, implying that this shoot signal does not affect their formation. Different repression curves of the two responses imply that the induction of transfer cells occurs after the enhancement of electron transfer across the plasma membrane rather than being causally linked. Similar to transfer cells, the formation of extra root hairs in the Arabidopsis mutant man1 was regulated by the iron concentration of the growth medium and was unaffected by interorgan signaling.     

Modulation of the root epidermal phenotype by hormones,inhibitors and iron regime

Patterning of epidermal cells is subject to genetic regulation but also influenced by environmental stimuli. To adapt to unfavorable environmental conditions plants have developed various mechanisms to increase the plasma membrane's surface area of epidermal root cells, for example through the formation of root hairs and differentiation of rhizodermal transfer cells. Mechanisms controlling cell fate speciation in the rhizodermis were investigated by application of hormones and hormone antagonists. In addition, the effect of Fe deficiency on root epidermal patterning and Fe(III)-reduction activity was examined. In the iron-hyperaccumulating pea mutants dgl and brz and in the Arabidopsis mutant man1 Fe(III)-reduction activity was found to be up-regulated under both high and low iron supply. In contrast, morphological responses such as the development of transfer cells and extranumerary root hairs was repressed by a high iron concentration in the external medium. All morphological responses can be mimicked by exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or the auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D). Conversely, Fe(III)-reduction rates were not influenced or only slightly affected by the hormone treatment. Application of inhibitors of ethylene synthesis, ethylene action or auxin transport was effective only in inhibiting the formation of extra root hairs, indicating that these hormones are not required for transfer cell formation or expression of Fe(III) reduction. These data suggest that the Fe reductase induced by iron stress does not depend on the formation of transfer cells and further imply separate regulatory pathways for the two responses. The data are compatible with a model in which root reduction activity is modulated by a shoot-borne signal coordinating iron uptake with the shoot demand, while the epidermal phenotype is primarily dependent on the intracellular iron concentration of root cells.     

Physiological responses and differential gene expression in Prunus rootstocks under iron deficiency conditions

Two Prunus rootstocks, the Myrobalan plum P 2175 and the interspecific peach-almond hybrid, Felinem, were studied to characterize their biochemical and molecular responses induced under iron-Deficient conditions. Plants of both genotypes were submitted to different treatments using a hydroponic system that permitted removal of Fe from the nutrient solution. Control plants were grown in 90 μM Fe (III)-EDTA, Deficient plants were grown in an iron free solution, and plants submitted to an Inductor treatment were resupplied with 180 μM Fe (III)-EDTA over 1 and 2 days after a period of 4 or 15 days of growth on an iron-free solution. Felinem increased the activity of the iron chelate reductase (FC-R) in the Inductor treatment after 4 days of iron deprivation. In contrast, P 2175 did not show any response after at least 15 days without iron. The induction of the FC-R activity in this genotype was coincident in time with the medium acidification. These results suggest two different mechanisms of iron chlorosis tolerance in both Strategy I genotypes. Felinem would use the iron reduction as the main mechanism to capture the iron from the soil, and in P 2175, the mechanism of response would be slower and start with the acidification of the medium synchronized with the gradual loss of chlorophyll in leaves. To better understand the control of these responses at the molecular level, the differential expression of PFRO2, PIRT1 and PAHA2 genes involved in the reductase activity, the iron transport in roots, and the proton release, respectively, were analyzed. The expression of these genes, estimated by quantitative real-time PCR, was different between genotypes and among treatments. The results were in agreement with the physiological responses observed.     

The arbuscular mycorrhizal fungus Rhizophagus irregularis uses a reductive iron assimilation pathway for high‐affinity iron uptake

Arbuscular mycorrhizal (AM) fungi can improve iron (Fe) acquisition of their host plants. Here, we report a characterization of two components of the high‐affinity reductive Fe uptake system of Rhizophagus irregularis, the ferric reductase (RiFRE1) and the high affinity Fe permeases (RiFTR1‐2). In the extraradical mycelia (ERM), Fe deficiency induced activation of a plasma membrane‐localized ferric reductase, an enzyme that reduces Fe(III) sources to the more soluble Fe(II). Yeast mutant complementation assays showed that RiFRE1 encodes a functional ferric reductase and RiFTR1 an iron permease. In the heterologous system, RiFTR1 was expressed in the plasma membrane while RiFTR2 was expressed in the endomembranes. In the ERM, the highest expression levels of RiFTR1 were found in mycelia grown in media with 0.045 mM Fe, while RiFTR2 was upregulated under Fe‐deficient conditions. RiFTR2 expression also increased in the intraradical mycelia (IRM) of maize plants grown without Fe. These data indicate that the Fe permease RiFTR1 plays a key role in Fe acquisition and that RiFTR2 is involved in Fe homeostasis under Fe‐limiting conditions. RiFTR1 was highly expressed in the (IRM), which suggests that the maintenance of Fe homeostasis in the IRM might be essential for a successful symbiosis.     

Excessive iron accumulation in the pea mutants dgl and brz: subcellular localization of iron and ferritin

The pea (Pisum sativum L.) mutants dgl and brz are defective in the regulation of iron uptake. Enhanced proton extrusion and constitutively high Fe(III) reductase activities in the roots lead to an accumulation of iron and other divalent cations in different organs of the mutants. Ultrastructural investigations of the basal leaflets of the mutants revealed in the cytoplasm, in mitochondria and especially in, or close to the endoplasmic reticulum numerous electron-dense particles which were absent in the corresponding wild type plants DGV and Sparkle. By means of electron-spectroscopic imaging and electron-energy-loss spectroscopy it could be shown that these electron-dense particles consist mainly of iron. Some of the iron deposits were immunocytochemically identified as the iron-storage protein ferritin. It is suggested that the precipitation of excessive iron in the dgl and brz mutant leaves in the form of electron-dense iron particles combined with the accumulation of ferritin is part of the plant defense mechanism against Fe-mediated oxidative stress. Received: 17 February 1998 / Accepted: 4 July 1998     

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.     

Zinc uptake by rice as affected by iron and a chelator of ferrous iron

Summary Iron competitively inhibited Zn absorption by rice (Oryza sativa L. cv. Earlirose) grown in solution culture. The effect was more marked for shoots since Fe had also a competitive effect on Zn translocation from roots to shoots. The chelating agent baptholphenanthrolinesulfonate (BPDS), which has great ability to chelate Fe⁺⁺, alleviated the inhibitory effect of Fe to a large extent. re]19750516     

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.     

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.     

Iron assimilation in Chlamydomonas reinhardtii involves ferric reduction and is similar to Strategy I in higher plants

The mechanism of adaptation to Fe-deficiency stress was investigated in the unicellular green alga, Chlamydomonas reinhardtii. Upon removal of nutritional Fe, the activity of a cell surface Fe(III)-chelate reductase was increased by at least 15-fold within 24 h. This increase was negatively corelated with the Fe concentration in the growth media. Incubation of cells in the presence of the Fe²⁺-specific chelator, bathophenanthrolinedisulphonic acid, led to an increased Fe³⁺ reductase activity, even when sufficient Fe was present. Growth of cells in Cu-free media for 48 h led to no statistically significant increase in Fe³⁺ reductase activity. The Fe(III)-chelate reductase activity in Fe-starved cells was saturable with an apparent Km of 31 M and was inhibited by uncouplers of the transmembrane proton gradient but not by SH-specific reagents.Fe uptake was only observed in Fe-deficient cells. Uptake was specific for Fe in that at 100-fold excess of a number of metal ions in the transport assay did not inhibit uptake activity. However, a 100-fold excess of Cu resulted in a 87% inhibition of Fe uptake. The Vmax for Fe³⁺ reduction activity was 250-fold greater than for Fe uptake; although the Km values for both processes differed by only 10-fold. Thus, the rate limiting step in Fe assimilation was transport and not reduction. These results indicate that Fe assimilation in C. reinhardtii involves a reductive step and thus resembles the mechanism of Fe uptake in Strategy I higher plants.Keywords: Ferric chelate reduction, iron assimilation, iron uptake, unicellular green algae, Chlamydomonas.      

不同pH值下丛枝菌根真菌对枳生长及铁吸收的影响

摘要：【目的】本文对营养液不同pH值下丛枝菌根(arbuscular mycorrhiza)真菌地表球囊霉（Glomus versiforme）对枳[Poncirus trifoliata]实生苗生长及植株铁营养状况的影响进行了初步研究。【方法】采用盆栽砂培试验,分别施浇pH 5.0、6.0、7.0和8.0的霍格兰营养液（含50 μM Fe-EDTA）;常规方法测定植株生长指标;曲利苯蓝染色法测定菌根侵染率;分光光度法测定叶绿素含量和根系三价铁螯合物还原酶活性;原子吸收分光光度法测定叶片钾和活性铁含量;钒     

Induction of iron(III) and copper(II) reduction in pea (Pisum sativum L.) roots by Fe and Cu status: Does the root-cell plasmalemma Fe(III)-chelate reductase perform a general role in regulating cation uptake?

We investigated the effects of Fe and Cu status of pea (Pisum sativum L.) seedlings on the regulation of the putative root plasma-membrane Fe(III)-chelate reductase that is involved in Fe(III)-chelate reduction and Fe²⁺ absorption in dicotyledons and nongraminaceous monocotyledons. Additionally, we investigated the ability of this reductase system to reduce Cu(II)-chelates as well as Fe(III)-chelates. Pea seedlings were grown in full nutrient solutions under control, -Fe, and -Cu conditions for up to 18 d. Iron(III) and Cu(II) reductase activity was visualized by placing roots in an agarose gel containing either Fe(III)-EDTA and the Fe(II) chelate, Na₂bathophenanthrolinedisulfonic acid (BPDS), for Fe(III) reduction, or CuSO₄, Na₃citrate, and Na₂-2,9-dimethyl-4,7-diphenyl-1, 10-phenanthrolinedisulfonic acid (BCDS) for Cu(II) reduction. Rates of root Fe(III) and Cu(II) reduction were determined via spectrophotometric assay of the Fe(II)-BPDS or the Cu(I)-BCDS chromophore. Reductase activity was induced or stimulated by either Fe deficiency or Cu depletion of the seedlings. Roots from both Fe-deficient and Cu-depleted plants were able to reduce exogenous Cu(II)-chelate as well as Fe(III)-chelate. When this reductase was induced by Fe deficiency, the accumulation of a number of mineral cations (i.e., Cu, Mn, Fe, Mg, and K) in leaves of pea seedlings was significantly increased. We suggest that, in addition to playing a critical role in Fe absorption, this plasma-membrane reductase system also plays a more general role in the regulation of cation absorption by root cells, possibly via the reduction of critical sulfhydryl groups in transport proteins involved in divalent-cation transport (divalent-cation channels?) across the root-cell plasmalemma.