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Proteins containing an iron–sulfur (Fe–S) cluster(s) as a prosthetic group play pivotal roles in diverse cellular processes, including respiration, photosynthesis, response to oxidative conditions, and genome stability (Rouault & Klausner, 1996; Balk & Pilon, 2011). Several Fe–S cluster biogenesis pathways exist in bacteria, such as the sulfur mobilization (SUF), iron–sulfur cluster (ISC) and nitrogen fixation (Nif) systems; these systems have also been found in different cellular compartments of eukaryotes (Lill & Mühlenhoff, 2006). The SUF machinery is conserved from archaea to chloroplasts in plants (Takahashi & Tokumoto, 2002; Godman & Balk, 2008), and mitochondrial ISC systems have been identified in eukaryotic cells (Lill & Mühlenhoff, 2005).
Several studies in yeast identified a novel Fe–S cluster assembly system, which has been named the cytosolic iron–sulfur cluster assembly (CIA) pathway (Lill, 2009). Unlike the SUF and ISC systems, the CIA system was found mainly in eukaryotes. This system is essential for the maturation of both cytosolic and nuclear Fe–S proteins (Lill & Mühlenhoff, 2008). For example, the yeast ATP-binding cassette transporters RNase L Inhibitor (Rli1) and isopropyl malate isomerase (Leu1p) are representative Fe–S proteins in the cytosol (Kispal et al., 1999, 2005), while many nuclear Fe–S proteins, such as an excision repair protein XPD helicase and DNA polymerase delta, are involved in DNA metabolism (Kispal et al., 1999, 2005; Rundolf et al., 2006). In addition to identification of many cytosolic and nuclear Fe–S proteins, recent studies have also found that the CIA pathway consists of multiple proteins. The cytosolic Fe–S cluster assembly factor Nuclear Architecture Related 1 (NAR1) seems to be responsible for incorporation of an Fe–S group into the apoprotein (Balk et al., 2004). Cytosolic Fe–S cluster Deficient 1 (Cfd1) and Nucleotide Binding Protein 35 (Nbp35) provide a scaffold for assembly of Fe–S clusters (Netz et al., 2007), and Top1T722A mutant Hypersensitive 18 (Tah18) and Derepressed for Ribosomal protein S14 Expression 2 (Dre2) are involved in electron-transfer processes in an early step of Fe–S cluster assembly in the cytoplasm (Netz et al., 2010). Interestingly, Nar1, Nbp35 and Dre2 proteins also contain binding motifs for Fe–S clusters (Balk et al., 2004; Hausmann et al., 2005; Zhang et al., 2008). The protein Cia1, which has a WD40 repeat motif, is believed to serve as a platform for protein–protein interactions in the CIA pathway (Balk et al., 2005). In addition, recent studies have reported that Cia2 (YHR122w) and Mms19 (gene YIL128w) proteins are components of the CIA pathway, required for DNA metabolism and maintenance of genome integrity (Gari et al., 2012; Stehling et al., 2012). Thus, over the last decade, remarkable progress has been made in elucidation of the yeast CIA pathway.
Associated with this recent progress in yeast, studies have begun to uncover the Fe–S cluster assembly pathway in plants and to determine its biological roles. Comparative genomic analyses in green algae and other species suggest that plants show conservation of proteins of the Fe–S cluster assembly pathway (Godman & Balk, 2008). According to these analyses, plant genomes do not have a Cfd1 homolog; instead, NBP35 seems to form a homodimer that is responsible for the necessary scaffold (Bych et al., 2008). Studies using recombinant homologs of the NAR1 and NBP35 proteins in Arabidopsis thaliana revealed that these proteins are able to coordinate the [4Fe–4S] cluster(s) (Bych et al., 2008; Cavazza et al., 2008; Kohbushi et al., 2009). In addition, deficiencies in NBP35 or Arabidopsis Thaliana P450 Reductase; ATR3 (the A. thaliana homolog of yeast Tah18) cause seed abortion (Bych et al., 2008; Cavazza et al., 2008; Kohbushi et al., 2009). A new component of the CIA pathway in A. thaliana has recently been reported: ASYMMETRIC LEAVES1/2 ENHANCER7 (AE7), which is probably the plant counterpart of CIA2, is required for activity of the Fe–S protein cytosolic aconitase; it is also essential to the proper activity of the nuclear Fe–S protein ROS1 (a DNA demethylase) based on the observation of hypermethylation of Repressor Of Silencing 1 (ROS1) target loci (Luo et al., 2012). However, much work still needs to be done to understand the physiological properties of the CIA pathway in higher plants throughout the life cycle.
Here, we isolated a nar1-3 mutant of the CIA pathway in A. thaliana and showed that it had impaired expression of FWA-GFP in the central cell and endosperm. FWA encodes an Homeodomain leucine zipper IV (HD-ZipIV) class homeodomain transcription factor. When the FWA gene is overexpressed in vegetative tissues of DNA methylation-deficient mutants (e.g. methyltransferase 1 (met1) and decreased DNA methylation 1 (ddm1)), it results in a late-flowering phenotype (Soppe et al., 2000; Kankel et al., 2003). However, in the wild-type, FWA is always silent during vegetative growth and is expressed specifically in endosperm tissue (Kinoshita et al., 2004). The FWA-GFP reporter mimics the imprinted gene expression pattern of endogenous FWA. This expression is controlled by the DNA glycosylase DEMETER (DME) (Kinoshita et al., 2004), which has a motif associated with an Fe–S cluster that is important for 5-methylcytosine excision activity (Mok et al., 2010). In addition, genetic analyses in the reproductive phase showed that NAR1 is required for both gametophytic and zygotic functions. We also determined the transcription profile of the weak nar1-4/− allele in vegetative tissues and compared this profile to that of nbp35-3/−, a mutant of a component of the Fe–S scaffold in the CIA pathway. The genetic analyses of these different nar1 alleles demonstrated that NAR1 is required for the transcriptional activation of some imprinted genes in female gametophytes and for proper abiotic stress responses in vegetative tissues.
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In view of the number of putative Fe–S proteins in the cytosol and nucleus, it would appear that the CIA pathway is essential to a wide range of cellular processes. Thus, detailed phenotypic analyses of mutant alleles of the component genes will provide valuable information on the roles of the CIA pathway. In this study, we characterized mutations of NAR1, a gene that encodes an Fe-hydrogenase-like protein that appears to function in the CIA pathway (Luo et al., 2012). Our analyses showed that NAR1 is required for the expression of the imprinted FWA gene and for seed development, and is involved in oxidative stress responses in the vegetative tissues.
A possible mechanism for transcriptional activation of endosperm-specific genes
The nar1-3 mutation was identified in a screen for impaired transcriptional activation of the endosperm-specific gene FWA. The CIA pathway seems to control gene transcription, as RNA polymerase subunits and transcription elongation factors have Fe–S cluster binding motifs (Hirata & Murakami, 2009; Okada et al., 2010). However, it is unlikely that NAR1 could influence global transcription as we found that MEA-GFP was normally activated in the central cell and endosperm (Fig. 3c,d). In the qRT-PCR analysis, the normal expression of MEA in seeds also excludes the possibility that impaired FWA and FIS2 expression was attributable to abnormal female gametophyte development (Fig. 2). Overall, our evidence suggests that impaired transcriptional activation of FWA and FIS2 in nar1 mutants is mediated through another more gene-specific system. FWA, FIS2 and MEA are genes known to be imprinted via changes in DNA methylation. However, the nar1 mutation did not affect MEA expression in the endosperm, in contrast to FWA and FIS2. This difference may be explained by the presence of an additional regulatory mechanism at the MEA locus (Baroux et al., 2006; Gehring et al., 2006; Jullien et al., 2006a,b). Therefore, the fact that the nar1 mutation affects FWA and FIS2 but not MEA implies that NAR1 may be involved in regulating gene expression simply via the DNA methylation pattern. It is notable that impaired FWA-GFP expression was observed in half of the ovules before fertilization (Fig. 1a,b). Additionally, an impaired FWA-GFP phenotype was evident even when wild-type pollen was used for pollination (Fig. 1c–f). These observations suggest that the impairment of transcriptional activation of FWA and FIS2 may be a maternal gametophytic effect, as both genes are known to be imprinted. Imprinted genes show a parent-of-origin effect on expression. It is known that transcriptional activation of many imprinted genes requires the DNA demethylase activity of DME. The DME protein has an Fe–S cluster loop motif (Stoesser et al., 2002). Given that DME functions in the nucleus, the protein is a possible target for the CIA pathway. Indeed, it was reported that A. thaliana plants homozygous for the ae7 gene mutation have defects in their CIA pathway and exhibit DNA hypermethylation at specific loci in the vegetative phase (Luo et al., 2012). Taken together, these data suggest that NAR1 could influence FWA-GFP expression through a DNA (de)methylation mechanism.
NAR1 is required not only for zygotic development but also for paternal transmission
Inactivation of two endosperm-specific genes, FWA and FIS2, in nar1-3 is attributable to a female gametophytic effect (Fig. 1a); by contrast, embryo defects were only observed when the nar1 heterozygous mutant was self-pollinated (Tables 1, 2). Most mutants of CIA components fail to generate homozygous mutants (Bych et al., 2008; Kohbushi et al., 2009; Varadarajan et al., 2010; Luo et al., 2012). We investigated the transmission rates and compared embryo phenotypes between nar1-3, nbp35-1, cia1-1 and atr3-3/tah18-3 alleles. This analysis showed that embryo lethality was a zygotic effect and that putative homozygous plants were aborted at an early embryo stage (Figs 3e–h, S4c–h, Tables 2, 3). A similar observation has been made in mice, where knockout of the NAR1 homolog IOP1 leads to lethality before embryonic day 10.5 (Song & Lee, 2008). Thus, CIA components are essential to early embryonic development in both plants and animals. Among the four CIA components investigated, nar1 alleles showed a partial male-specific transmission defect (Table 3). In this respect, therefore, NAR1 appears to be different from the other CIA components. In yeast studies, each component showed a somewhat different cellular localization pattern or different maturation process (Balk et al., 2004, 2005; Hausmann et al., 2005; Vernis et al., 2009). These data may suggest that each CIA pathway component functions at a different step of the pathway and not as one complex. This might explain why not all of the already characterized CIA mutants share all the phenotypes. To our knowledge, the additional role of NAR1 in the male gametophyte is first reported here.
The role of NAR1 in iron homeostasis and oxidative stress
To circumvent embryonic lethality and gain further insights into the role of the CIA pathway in vegetative tissues, we selected the weak alleles nar1-4 and nbp35-3 and used these to produce viable homozygotes. A transcriptional profile of nar1-4/− showed that five genes involved in ‘iron ion transport’ and ‘response to nitrate’ were down-regulated in the homozygote mutant. This may reflect a physiological compensation in nar1-4 through iron homeostasis. Previous reports indicate that nitric oxide targets some Fe–S proteins and regulates their activities catalytically or structurally (Butler & Megson, 2002). Therefore, if nitric oxide signaling is mediated through Fe–S proteins, it is possible that defects in the Fe–S cluster assembly in nar1 might cause an impaired response to these signals. Further analyses are necessary to investigate these possibilities. Iron is used in the form of Fe–S clusters or heme in organisms. The dysregulation of iron/sulfur homeostasis might therefore be a source of oxidative stress, indicating that organisms may need to strictly regulate this homeostasis in cellular processes. Hence, differentially expressed genes in seedlings seem to reflect the defects in iron homeostasis in the nar1-4 mutant rather than a specific transcriptional regulation (Table S3).
The Fe–S clusters of some proteins are sensitive to oxidative damage (Crack et al., 2012). The molecular mechanism for resistance to paraquat in nar1-4/− is unknown, but one possibility is that the generation of reactive oxygen species or the induction of the cell death pathway might be influenced by a defective Fe–S protein(s). Interestingly, yeast mutants for another CIA component, Tah18, show resistance to H2O2, an oxidative stress reagent (Vernis et al., 2009). Vernis et al. postulated that the yeast Tah18 protein might be required for cell death signaling to mitochondria. An alternative explanation comes from the report that a plant with decreased aconitase activity showed a reduction in paraquat sensitivity (Moeder et al., 2007). Reduced aconitase activity in nar1-4/− supports the idea that the failure of Fe–S cluster incorporation into aconitase proteins in the absence of the intrinsic CIA pathway leads to paraquat resistance in nar1-4/−.