Iron deficiency decreases the Fe(III)-chelate reducing activity of leaf protoplasts

The ferric-chelate reductase (FC-R) activity of mesophyll protoplasts isolated from Fe-sufficient (control) and Fe-deficient sugar beet (Beta vulgaris L.) leaves has been characterized. Measurements were made in an ionic environment similar to that in the apoplastic space of the sugar beet mesophyll cells. The FC-R activity of Fe-sufficient and Fe-deficient protoplasts was dependent on light. Fe deficiency decreased markedly the FC-R activity per protoplast surface unit. The optimal pH for the activity of the FC-R in mesophyll protoplasts was in the range 5.5 to 6.0, typical of the apoplastic space. Beyond pH 6.0, the activity of the FC-R in mesophyll protoplasts decreased markedly in both Fe-sufficient and Fe-deficient protoplasts. These data suggest that both the intrinsic decrease in FC-R activity per protoplast surface and a possible shift in the pH of the apoplastic space could lead to the accumulation of physiologically inactive Fe pools in chlorotic leaves.     

Changes in sugar beet leaf plasma membrane Fe(III)-chelate reductase activities mediated by Fe-deficiency, assay buffer composition, anaerobiosis and the presence of flavins

Summary Different assay conditions induce changes in the ferric chelate reductase activities of leaf plasma membrane preparations from Fe-deficient and Fe-sufficient sugar beet. With an apoplasttype assay medium the ferric chelate reductase activities did not change significantly when Fe(III)-EDTA was the substrate. However, with ferric citrate as substrate, the effect depended on the citrateto-Fe ratio. When the citrate-to-Fe ratio was 20 1, the effects were practically unappreciable. However, with a lower citrate-to-Fe ratio of 5 1 the activities were significantly lower with the apoplast-type medium than with the standard assay medium. Our data also indicate that anaerobiosis during the assay facilitates the reduction of ferric malate and Fe(III)-EDTA by plasma membrane preparations. Anaerobiosis increased by approximately 50% the plasma membrane ferric chelate reductase activities when Fe(III)-EDTA was the substrate. With ferric malate anaerobiosis increased activities by 70–90% over the values obtained in aerobic conditions. However, with ferric citrate the increase in activity by anaerobiosis was not significant. We have also tested the effect of riboflavin, flavin adenine dinucleotide, and flavin mononucleotide on the plasma membrane ferric chelate reductase activities. The presence of flavins generally increased activities in plasma membrane preparations from control and Fe-deficient plants. Increases in activity were generally moderate (lower than twofold). These increases occurred with Fe(III)-EDTA and Fe(III)-citrate as substrates.Abbreviations BPDS bathophenantroline disulfonate - FC ferric chelate - FC-R ferric chelate reductase - PM plasma membrane     

Iron requirement for and effects of promoters and inhibitors of ethylene action on stimulation of Fe(III)-chelate reductase in roots of strategy I species

Stimulation of root Fe(III) reductase activity by iron additions to iron-deficient growth media may be the result of iron activation of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase required for ethylene biosynthesis. Two different ethylene inhibitors, aminooxyacetic acid (AOA) (20 m; ACC synthase inhibitor) and cobalt (3 m CoCl₂; ACC oxidase inhibitor), were used to study the effects of iron supply and cobalt inhibition on ethylene action in controlling the activity of Fe(III)-chelate reductase in pea (Pisum sativum L.) roots. Supplying 20 gm m Fe(III)-N,N-ethylenebis[2-(2-hydroxypheyl)-glycine [Fe(III)-EDDHA] to either cobalt-treated, iron-deficient Sparkle (normal parent) or E107 (brz mutant genotype) pea seedlings reversed the negative effects of cobalt on root Fe(III)-reductase activity. Re-supplying 20 m Fe(III)-EDDHA to iron-deficient, AOA-treated seedlings did not enhance root Fe(III)-reductase. Apparently, cobalt competes with iron for the active site in ACC oxidase during ethylene synthesis. Inhibition of root reductase activity by cobalt treatment lowered manganese, zinc, magnesium and potassium content of mutant E107 pea seedlings. In contrast, iron enhancement of root reductase activity in iron-deficient, cobalt-treated E107 seedlings resulted in higher seedling accumulations of manganese, zinc, magnesium and potassium. These results support the hypothesis that root cell plasma membrane reductase activity plays a role in cation uptake by root cells.     

Oxidative stress,antioxidant activity and Fe(III)-chelate reductase activity of five <Emphasis Type=Italic>Prunus</Emphasis> rootstocks explants in response to Fe deficiency

The aim of this work was to investigate whether Fe reduction and antioxidant mechanisms were expressed differently in five Prunus rootstocks (‘Peach seedling,’ ‘Barrier,’ ‘Cadaman,’ ‘Saint Julien 655/2’ and ‘GF-677’). These rootstocks differ in their tolerance to Fe deficiency when grown in the absence of Fe (−Fe) or in presence of bicarbonate (supplied as 5 or 10 mM NaHCO₃). Fe deficiency conditions, especially bicarbonate, were shown to decrease Fe and total chlorophyll (CHL) concentration. In the (−Fe)-treated roots of all rootstocks and in the 5 mM NaHCO₃-treated ones of the tolerant ‘GF-677’ the Fe(III)-chelate reductase (FCR) activity was stimulated. On the contrary, apart from the ‘GF-677,’ FCR activity was greatly inhibited by the 10 mM NaHCO₃. From the results obtained with decapitated rootstocks, it is not entirely clear whether or not the presence of shoot apex was a prerequisite to induce FCR function in all rootstocks tested. In the leaves of rootstocks exposed to the (−Fe) treatment, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities were enhanced whereas the levels of the non-enzymatic antioxidants (FRAP values) were increased in the Fe-deprived leaves, irrespective of the rootstock. Except for ‘Peach seedling,’ foliar SOD activity was stimulated by the presence of NaHCO₃. Furthermore, POD activity was increased in the ‘Saint Julien 655/2’ and ‘GF-677,’ but was depressed in the ‘Barrier’ rootstocks exposed to 10 mM NaHCO₃. As a result of 10 mM NaHCO₃, the expression of a Cu/Zn-SOD and a POD isoform was diminished in the leaves of ‘Peach seedling’ and ‘Barrier,’ respectively. By contrast, an additional isoform of both POD and Mn–SOD were expressed in the leaves of ‘GF-677’ exposed to 10 mM NaHCO₃ suggesting that the tolerance of rootstocks to Fe deficiency is associated with induction of an antioxidant defense mechanism. Although CAT activity was increased in the 5 mM NaHCO₃-treated leaves of ‘GF-677,’ specifically the 10 mM NaHCO₃ treatment resulted in a decrease of CAT activity and an accumulation of H₂O₂, indicating that bicarbonate-induced Fe deficiency may cause more severe oxidative stress in the rootstocks, than the absence of Fe. A general link between Fe deficiency-induced oxidative stress and Fe reduction-sensing mechanism is also discussed.     

Isolation of a soluble and membrane-associated Fe(III) reductase from the thermophile, Thermus scotoductus (SA-01)

The Fe(III) reductase activity was studied in the South African Fe(III)-reducing bacterium, Thermus scotoductus (SA-01). Fractionation studies revealed that the membrane as well as the soluble fraction contained NAD(P)H-dependent Fe(III) reductase activity. The membrane-associated enzyme was solubilized by KCl treatment and purified to electrophoretic homogeneity by hydrophobic interaction chromatography. A combination of ion-exchange and gel filtration chromatography was used to purify the soluble enzyme to apparent homogeneity. The molecular mass of the membrane-associated Fe(III) reductase was estimated to be 49 kDa, whereas the soluble Fe(III) reductase had an apparent molecular mass of 37 kDa. Optimum activity for the membrane-associated enzyme was observed at around 75 degrees C, whereas the soluble enzyme exhibited a temperature optimum at 60 degrees C.     

AtHVA22 gene family in Arabidopsis: phylogenetic relationship,ABA and stress regulation,and tissue-specific expression

HVA22 is an ABA- and stress-inducible gene first isolated from barley (Hordeum vulgare L.). Homologues of HVA22 have been found in plants, animals, fungi and protozoa, but not in prokaryotes, suggesting that HVA22 plays a unique role in eukaryotes. Five HVA22 homologues, designated AtHVA22a, b, c, d and e, have been identified in Arabidopsis. These five AtHVA22 homologues can be separated into two subfamilies, with AtHVA22a, b and c grouped in one subfamily and AtHVA22d and e in the other. Phylogenetic analyses show that AtHVA22d and e are closer to barley HVA22 than to AtHVA22a, bandc, suggesting that the two subfamilies had diverged before the divergence of monocots and dicots. The distribution and size of exons of AtHVA22 homologues and barley HVA22 are similar, suggesting that these genes are descendents of a common ancestor. AtHVA22 homologues are differentially regulated by ABA, cold, dehydration and salt stresses. These four treatments enhance AtHVA22a, d and e expression, but have little or even suppressive effect on AtHVA22c expression. ABA and salt stress induce AtHVA22b expression, but cold stress suppresses ABA induction of this gene. Expression of AtHVA22d is the most tightly regulated by these four treatments among the five homologues. In general, AtHVA22 homologues are expressed at a higher level in flower buds and inflorescence stems than in rosette and cauline leaves. The expression level of these homologues in immature siliques is the lowest among all tissues analyzed. It is suggested that some of these AtHVA22 family members may play a role in stress tolerance, and others are involved in plant reproductive development.     

AtHVA22 gene family in Arabidopsis: phylogenetic relationship,ABA and stress regulation,and tissue-specific expression

HVA22 is an ABA- and stress-inducible gene first isolated from barley (Hordeum vulgare L.). Homologues of HVA22 have been found in plants, animals, fungi and protozoa, but not in prokaryotes, suggesting that HVA22 plays a unique role in eukaryotes. Five HVA22 homologues, designated AtHVA22a, b, c, d and e, have been identified in Arabidopsis. These five AtHVA22 homologues can be separated into two subfamilies, with AtHVA22a, b and c grouped in one subfamily and AtHVA22d and e in the other. Phylogenetic analyses show that AtHVA22d and e are closer to barley HVA22 than to AtHVA22a, b and c, suggesting that the two subfamilies had diverged before the divergence of monocots and dicots. The distribution and size of exons of AtHVA22 homologues and barley HVA22 are similar, suggesting that these genes are descendents of a common ancestor. AtHVA22 homologues are differentially regulated by ABA, cold, dehydration and salt stresses. These four treatments enhance AtHVA22a, d and e expression, but have little or even suppressive effect on AtHVA22c expression. ABA and salt stress induce AtHVA22b expression, but cold stress suppresses ABA induction of this gene. Expression of AtHVA22d is the most tightly regulated by these four treatments among the five homologues. In general, AtHVA22 homologues are expressed at a higher level in flower buds and inflorescence stems than in rosette and cauline leaves. The expression level of these homologues in immature siliques is the lowest among all tissues analyzed. It is suggested that some of these AtHVA22 family members may play a role in stress tolerance, and others are involved in plant reproductive development.     

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.     

Cloning and expression of 5, 10-Methylenetetrahydrofolate reductase (MTHFR) gene

Hyperhomocysteinemia is an independent risk factor of cardiovascular diseases and birth defects. One of the important factors causing hyperhomocysteinemia is decrease of 5,10-Methylenetetrahydrofdate reductase. Human and rat MTHFR cDNAs with RT-PCR were isolated, a prokaryodytic expression vector containing human MTHFR cDNA was constructed, and human MTHFR protein was expressed inE. coli. It was also found that the expression of rat MTHFR could be promoted by IL-1 and homocysteine.     

Gene duplication, tissue-specific gene expression and sexual conflict in stalk-eyed flies (Diopsidae)

Gene duplication provides an essential source of novel genetic material to facilitate rapid morphological evolution. Traits involved in reproduction and sexual dimorphism represent some of the fastest evolving traits in nature, and gene duplication is intricately involved in the origin and evolution of these traits. Here, we review genomic research on stalk-eyed flies (Diopsidae) that has been used to examine the extent of gene duplication and its role in the genetic architecture of sexual dimorphism. Stalk-eyed flies are remarkable because of the elongation of the head into long stalks, with the eyes and antenna laterally displaced at the ends of these stalks. Many species are strongly sexually dimorphic for eyespan, and these flies have become a model system for studying sexual selection. Using both expressed sequence tag and next-generation sequencing, we have established an extensive database of gene expression in the developing eye-antennal imaginal disc, the adult head and testes. Duplicated genes exhibit narrower expression patterns than non-duplicated genes, and the testes, in particular, provide an abundant source of gene duplication. Within somatic tissue, duplicated genes are more likely to be differentially expressed between the sexes, suggesting gene duplication may provide a mechanism for resolving sexual conflict.     

Fe(III) mineral formation and cell encrustation by the nitrate-dependent Fe(II)-oxidizer strain BoFeN1

Understanding the mechanisms of anaerobic microbial iron cycling is necessary for a full appreciation of present‐day biogeochemical cycling of iron and carbon and for drawing conclusions about these cycles on the ancient Earth. Towards that end, we isolated and characterized an anaerobic nitrate‐dependent Fe(II)‐oxidizing bacterium from a freshwater sediment. The 16SrRNA gene sequence of the isolated bacterium (strain BoFeN1) places it within the β‐Proteobacteria, with Acidovorax sp. strain G8B1 as the closest known relative. During mixotrophic growth with acetate plus Fe(II) and nitrate as electron acceptor, strain BoFeN1 forms Fe(III) mineral crusts around the cells. The amount of the organic cosubstrate acetate present seems to control the rate and extent of Fe(II) oxidation and the viability of the cells. The crystallinity of the mineral products is influenced by nucleation by Fe minerals that are already present in the inoculum.     

Molecular organization and tissue-specific expression of an Arabidopsis 14-3-3 gene.

The 14-3-3 proteins, originally described as mammalian brain proteins, are ubiquitous in eukaryotes. We isolated an Arabidopsis 14-3-3 gene, designated GRF1-GF14 chi (for general regulatory factor1-G-box factor 14-3-3 homolog isoform chi), and characterized its expression within plant tissues. Sequence comparison of the GRF1-GF14 chi genomic clone with other 14-3-3 proteins demonstrated that the extreme conservation of 14-3-3 residues in several domains is encoded by the first three exons. The highly variable C-terminal domain is encoded by a divergent fourth exon that is unique among 14-3-3 homologs, suggesting that exon shuffling might confer gene-specific functions among the isoforms. The anatomical distribution and developmental expression of the Arabidopsis 14-3-3 protein were examined in transgenic plants carrying a GRF1-GF14 chi promoter-beta-glucuronidase construct. GF14 chi promoter activity was observed in the roots of both seedlings and mature plants. In immature flowers, GF14 chi promoter activity was localized to the buds. However, as the flowers matured, GF14 chi promoter activity was restricted to the stigma, anthers, and pollen. In immature siliques, GF14 chi promoter activity was initially localized to styles and abscission zones but was subsequently observed throughout mature siliques. In situ hybridization demonstrated that GF14 chi mRNA expression was prominent in epidermal tissue of roots, petals, and sepals of flower buds, papillae cells of flowers, siliques, and endosperm of immature seeds. Thus, plant 14-3-3 gene expression exhibits cell- and tissue-specific localization rivaling that observed for 14-3-3 proteins within the mammalian brain.