The rice mitochondrial iron transporter is essential for plant growth

In plants, iron (Fe) is essential for mitochondrial electron transport, heme, and Fe-Sulphur (Fe-S) cluster synthesis; however, plant mitochondrial Fe transporters have not been identified. Here we show, identify and characterize the rice mitochondrial Fe transporter (MIT). Based on a transfer DNA library screen, we identified a rice line showing symptoms of Fe deficiency while accumulating high shoot levels of Fe. Homozygous knockout of MIT in this line resulted in a lethal phenotype. MIT localized to the mitochondria and complemented the growth of Δmrs3Δmrs4 yeast defective in mitochondrial Fe transport. The growth of MIT-knockdown (mit-2) plants was also significantly impaired despite abundant Fe accumulation. Further, the decrease in the activity of the mitochondrial and cytosolic Fe-S enzyme, aconitase, indicated that Fe-S cluster synthesis is affected in mit-2 plants. These results indicate that MIT is a mitochondrial Fe transporter essential for rice growth and development

Frataxin, a protein involved in iron metabolism, in Arabidopsis thaliana

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Ferritins and frataxin take part in the iron homeostasis and trafficking in Arabidopsis mitochondria

Although iron has a major role in mitochondrial activity, the processes of iron transport, trafficking and homeostasis in plant mitochondria are still mostly unknown. Ferritins are iron-storage proteins which, in plant cells, are localized in chloroplast but can also be localized in mitochondria. We characterized two independent Arabidopsis mutants, KO in the AtFer4 ferritin isoform. When grown in suspension cultures, atfer4 mutants show higher cell death and higher O2 consumption rates. Mitochondria of atfer4 cells are larger size and possess higher total iron content than wt ones, both in control conditions or upon treatment with iron excess. A more pronounced accumulation of a still unidentified ferritin(s) is observed in mitochondria of iron-treated atfer4 cells, when compared to wt ones. These results indicate that AtFer4 has a role for the iron trafficking/homeostasis in mitochondria in conditions in which such organelles are the sole energy source. Friedreich ataxia (FRDA), an autosomal recessive neurological dysfunction that severely impairs motor coordination in humans, is caused by a deficiency in frataxin, a nuclear-encoded mitochondrial protein. Although several groups have shown in the past that frataxin is involved in the assembly, regulation and repair of the [Fe-S] cluster, as well as in the homeostasis of mitochondrial iron, its primary role is still matter of debate. We have shown that the Arabidopsis frataxin homolog, named AtFH, is expressed mainly in flowers and developing embryos and that it encodes an essential protein, since the knocking out of AtFH gene causes arrest of Arabidopsis embryo development at the globular stage. To overcome experimental constraints due to embryo lethality of AtFH KO, we are currently producing Arabidopsis transgenic lines, by inducible RNA-interference, in which microRNA can switch off AtFH frataxin transcript accumulation upon treatment with either ethanol or dexametasone

Knockout of frataxin causes embryo lethality in Arabidopsis

Frataxin is a nuclear encoded protein targeted to mitochondrial matrix. In humans, frataxin deficiency is associated with Friedrich\u2019s ataxia, a neurodegenerative and cardiac disorder characterized by accumulation of iron in mitochondria and a diminished activity of various mitochondrial proteins. It has been proposed that frataxin acts as chaperone to assist assembly of [Fe-S] clusters and heme synthesis. Recently, an Arabidopsis gene (AtFH) highly similar to the human frataxin gene and possessing a mitochondrial target domain has been described. Our preliminary RT-PCR analysis of AtFH transcript aboundance shows that AtFH transcript accumulate in cell cultures as well as in various plants organs, and in particular in flowers. However cells treated with excess iron do not accumulate higher AtFH transcript levels than wt. To understand physiological role of frataxin in plants, we identified two independent Arabidopsis heterozygous AtFH knockout mutants (from the Salk collection of insertional mutants); both of them have T-DNA inserted in the AtFH coding region. No homozygous KO lines could be recovered in any of the progeny obtained by selfing the two different mutants. Also, progeny obtained from selfing heterozygous plants segregated with 1 homozygous wt: 2 heterozygous KO ratio.These results are consistent with the hypothesis of lethality of the homozygous null genotypes during embryogenesis. Accordingly, pattern of embryo development in siliques segregating the putative homozygous null embryos show \ubc of all embryos arresting early at 8-16 cell stage. Finally, preliminary in situ hybridization shows accumulation of AtFH transcript in developing wt embryos. Taken together, these results suggest a role of frataxin during embryo development

Frataxin null mutants of arabidopsis are embryo lethals

Frataxin is a nuclear encoded protein targeted to the mitochondrial matrix. In humans, frataxin deficiency is associated with Friedreich\u2019s ataxia, a neurodegenerative and cardiac disorder characterized by accumulation of iron in the mitochondria and a diminished activity of various mitochondrial proteins, including aconitase. Yeast cells lacking frataxin show a complex respiratory deficient phenotype, defective in the maturation of mitochondrial Fe/S enzymes, hypersensitivity to oxidative stress, instability of mtDNA and defects in heme biosynthesis. It has been proposed that frataxin has ferroxidase activity and iron storage properties which may protect the mitochondria from iron toxicity, and that it also acts as a chaperone to donate iron to the proteins involved in the two major pathways of iron utilization, Fe/S cluster assembly and heme synthesis. Recently, an Arabidopsis gene (AtFH) highly similar to the human frataxin gene and possessing a mitochondrial targeting sequence has been described (Busi et al. 2004): AtFH is able to complement a yeast frataxin null mutant and in the plant is mainly expressed in flowers. We identified in the Salk collection two T-DNA insertions in the AtFH gene and characterized genetically the two mutants. Upon selfing heterozygous plants, we cannot recover in the progeny homozygote null seeds while homozygous wt and heterozygous seeds were in a ratio 1: 2 (as observed after PCR analysis). The ratio was consistent with lethality of the homozygous null genotypes during embryogenesis. Accordingly we analyze the pattern of embryo development in siliques segregating homozygous null embryos: an early arrest at the 8-16 cells stage was consistently observed

MOESM1 of Therapeutic efficacy of chloroquine for the treatment of Plasmodium vivax malaria among outpatients at Hossana Health Care Centre, southern Ethiopia

Additional file 1. WHO haemoglobin threshold used to define anaemia in different age and gender groups [19]

The proton pump interactor (Ppi) gene family of Arabidopsis thaliana : expression pattern of Ppi1 and characterisation of knockout mutants for Ppi1 and 2

Plant plasma membrane H+-ATPases (PM H+-ATPase) are essential for establishing a proton electrochemical gradient across the cell plasma membrane. Their regulation is poorly understood, except for the role of 14-3-3 proteins, which relieve autoinhibition from the C-terminal domain. A novel protein interacting with this domain was recently identified in Arabidopsis and named PPI1 (Proton Pump Interactor 1). PPI1 stimulates PM H+-ATPase activity in vitro. Here, we analyse the expression pattern of Ppi1 using beta-glucuronidase as a reporter. Expression is strong in root and shoot vascular systems, particularly in meristematic and sink tissues, as well as in pollen, stigmas and siliques, but not in developing embryos. Removal of the first intron decreased GUS expression 45-fold. We also analysed the transcription of Ppi2, another gene in the family, and demonstrated that Ppi2 is expressed in seedlings, cultured cells and flowers. We reassessed Ppi2 gene structure based on RT-PCR amplifications, cDNA data and similarity to other Ppi genes. Insertional mutants for both Ppi1 and Ppi2 were isolated. Two different mutants of Ppi1 showed aberrant mRNAs and lacked any detectable protein and are therefore true knockouts. Interestingly, one mutation inhibited the splicing of one intron at a considerable distance (>700 bp) from the T-DNA insertion site, but not the splicing of a proximal intron (29 bp) or of any other intron. At the plant level, neither of the single mutants nor the double ppi1ppi2 mutant showed an altered phenotype in standard growth conditions under acid load or salt stres

THE ATFER4 FERRITIN IS A KEY COMPONENT FOR THE CONTROL OF THE IRON HOMEOSTASIS IN MITOCHONDRIA OF ARABIDOPSIS CELLS.

Although iron has a major role in mitochondrial activity, the processes of iron transport, trafficking and homeostasis in plant mitochondria are mostly unknown. A first evidence of accumulation of the iron storage protein ferritin in plant mitochondria was recently shown but, up to now, the Arabidopsis ferritin gene encoding the mitochondrial isoform was not identified. According to computer analysis with four different programs (Target P, ChloroP, Mitoprot II, Predotar) predicting localization of protein in the different organelles, AtFer4 is the ferritin isoform with highest scores for localization in mitochondria. For this reason we therefore isolated and characterized two independent Arabidopsis atfer4 mutants knock out in the AtFer4 isoform, named atfer4-1 and atfer4-2. Analysis of atfer4-1 and atfer4-2 plant phenotypes was performed during seedling development, at mature plant stage and during senescence, in the aerial organs and in the roots: atfer4-1 and atfer4-2 mutant plants show no alteration of phenotype attributable to lack of AtFer4, at any stage or in any organ analysed, not even during senescence, which on the reverse is accelerated in atfer1 KO mutants. However, when grown in suspension cultures, atfer4 mutants show impaired growth and higher cell death. Purification of mitochondria from atfer4 suspension cultured cells allowed to show that ATFER4 is mitochondria-localized and that it accumulates in mitochondria regardless of exogenous iron treatment. Furthermore, at least another ferritin isoform, different from ATFER4, accumulates in mitochondria of iron-treated atfer4 and wt Col cells. Such accumulation at same levels in both atfer4 and wt Col mitochondria takes place, in atfer4 cells, under unexpected reduced accumulation of AtFer1, AtFer3 and AtFer2 transcripts. Interestingly, atfer4 mitochondria accumulate more iron than wt ones, both in control conditions or upon iron treatment. All these results indicate that ferritin targeting to mitochondria is a complex event involving constitutively ATFER4 and at least one other ferritin isoform under iron loading; also, they indicate that AtFer4 has a key role for the iron trafficking/homeostasis in mitochondria in conditions in which such organelles are the sole energy source, that is in suspension cultured cells

Characterization of knock out mutants in the PPI gene family : a T-DNA insertion causing aberrant splicing at distance

PPI1 (PROTON PUMP INTERACTOR 1) is a protein identified in a two hybrid screen whose N-terminus binds to the Arabidopsis thaliana plasma membrane proton pump (PM H+-ATPase). When added to purified membranes of Arabidopsis, the entire PPI1 protein, or fragments thereof, expressed as fusion protein in E. coli, are able to stimulate the activity of the proton pump (Morandini et al., 2002 Plant J. 31:487-97). To study the function of the PPI1 protein we make use of Arabidopsis lines bearing a T-DNA inserted in the gene. Two lines were characterized in detail for Ppi1: an insertion in intron I (named N93) and one in exon VII (F09) at aa 525. RT-PCR analysis on leaves and cell culture tissue confirmed that the transcript is aberrant in both insertion lines. In N93 homozygous plants, a low level of transcript is detectable, but the distal 5\u2019 UTR, normally present in the mRNA, is missing. In the case of F09 insertion, the message runs into the T-DNA region and presents premature terminations codons. Surprisingly a new, larger RNA molecule accumulates in this KO line because the splicing of the V intron is extensively prevented. The presence of the intron in the new RNA molecule was confirmed by PCR and by direct sequencing. The presence of premature termination codons causes similar phenomena in animal cells (Wang et al., 2002 Mol Cell 10:951-7). Western analysis performed on extracts from homozygous knock-out plants and from culture cells using an anti-PPI1 serum, confirms that the protein is absent in both insertion lines. In contrast, the protein as well the mRNA are present in the early stages of growth of wt seedlings and in leaves. In an alternative approach to study the pattern of Ppi1 expression, we also produced reporter constructs with the GUS gene under the control of Ppi1 promoter. Since the gene presents a large intron before the beginning of the coding region (a so-called \u2018leader intron\u2019), we decided to test three different constructs. The first contains both the promoter and the leader intron; the second one is lacking the leader intron but still contains with the Ppi1 5\u2019UTR; the last one is missing the intron and the 5\u2019UTR derives from the TMV. The Ppi1 KO lines do not show evident phenotype when grown in pots. Different growth conditions are being tested for identifying phenotypic differences in respect to wt. Two further insertion lines in Ppi3 and one in Ppi4 are being characterized at the molecular level