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. 2006 Jan 26;25(4):900–909. doi: 10.1038/sj.emboj.7600968

AtSufE is an essential activator of plastidic and mitochondrial desulfurases in Arabidopsis

Xiang Ming Xu 1, Simon Geir Møller 1,2,a
PMCID: PMC1383551  PMID: 16437155

Abstract

Iron–sulfur (Fe–S) clusters are vital prosthetic groups for Fe–S proteins involved in fundamental processes such as electron transfer, metabolism, sensing and signaling. In plants, sulfur (SUF) protein-mediated Fe–S cluster biogenesis involves iron acquisition and sulfur mobilization, processes suggested to be plastidic. Here we have shown that AtSufE in Arabidopsis rescues growth defects in SufE-deficient Escherichia coli. In contrast to other SUF proteins, AtSufE localizes to plastids and mitochondria interacting with the plastidic AtSufS and mitochondrial AtNifS1 cysteine desulfurases. AtSufE activates AtSufS and AtNifS1 cysteine desulfurization, and AtSufE activity restoration in either plastids or mitochondria is not sufficient to rescue embryo lethality in AtSufE loss-of-function mutants. AtSufE overexpression induces AtSufS and AtNifS1 expression, which in turn leads to elevated cysteine desulfurization activity, chlorosis and retarded development. Our data demonstrate that plastidic and mitochondrial Fe–S cluster biogenesis shares a common, essential component, and that AtSufE acts as an activator of plastidic and mitochondrial desulfurases in Arabidopsis.

Keywords: Arabidopsis , desulfurization, iron–sulfur cluster formation, mitochondria, plastids

Introduction

Iron–sulfur (Fe–S) clusters represent important and versatile prosthetic groups of Fe–S proteins involved in processes such as electron transport, redox and non-redox catalysis, sensing, signalling, DNA repair and regulation of gene expression (Beinert and Kiley, 1999; Balk and Lobreaux, 2005; Lill and Muhlenhoff, 2005). Although simple in structure, Fe–S biosynthesis requires the interplay of numerous proteins (Lill and Kispal, 2000; Frazzon and Dean, 2003; Balk and Lobreaux, 2005).

Fe–S cluster biogenesis is divided into elemental sulfur formation, iron acquisition, assembly of sulfur and iron into a cluster, and cluster insertion into apoproteins (Johnson et al, 2005; Lill and Muhlenhoff, 2005). Most research has come from bacteria which contain three main systems termed nitrogen fixation (NIF), iron–sulfur cluster (ISC) and mobilization of sulfur (SUF) (Balk and Lobreaux, 2005). Initial research came from studies in Azetobacter vinelandii (Frazzon and Dean, 2003), where the nitrogenase requires Fe–S cluster assembly by the cysteine desulfurase NifS. The NIF system is specifically involved only in the assembly and maturation of nitrogenase Fe–S clusters (Jacobson et al, 1989). The ISC system is more general and isc operon (iscSUA-hscBA-fdx) mutations in Escherichia coli result in decreased activities of numerous Fe–S proteins (Zheng et al, 1998; Schwartz et al, 2000; Tokumoto and Takahashi, 2001). In contrast to the NIF system, ISC components have been found in bacteria and higher eukaryotes (Muhlenhoff and Lill, 2000; Balk and Lobreaux, 2005).

The suf operon (sufABCDSE) represents the third Fe–S system, which can partially complement the isc operon (Takahashi and Tokumoto, 2002). SufB, SufC and SufD are conserved proteins, and in bacteria the cytosolic SufC ATPase interacts with SufB and SufD, presumably acting as an energizer for iron acquisition during Fe–S assembly (Loiseau et al, 2003; Nachin et al, 2003). Further, SufE interacts with SufS, where SufE accepts sulfur mobilized from cysteine and SufE/SufS may form a two-component cysteine desulfurase (Loiseau et al, 2003; Outten et al, 2003). Once acquired, SufA can assemble Fe–S clusters transiently in vitro (Ollagnier-de-Choudens et al, 2003).

In yeast and mammals all Fe–S clusters are thought to be generated in mitochondria, although this is still debated (Lill and Kispal, 2000; Rouault and Tong, 2005). In plants there is evidence that mitochondria harbor an Fe–S cluster biogenesis system involving the ABC transporter Sta1 and the putative cysteine desulfurase AtNifS1 (AtNSF1) (Kushnir et al, 2001). Indeed, the potato mitochondrial NifS homolog can activate biotin synthase (Picciocchi et al, 2003). The existence of the Arabidopsis chloroplast-localized Nif-like protein AtCpNIFS/AtNFS2/AtSufS and mitochondrial and plastidic NFU Fe–S cluster biogenesis proteins demonstrates that Fe–S cluster assembly in plants involves both mitochondria and plastids (Leon et al, 2002, 2003; Pilon-Smits et al, 2002).

Recently, homologs of the bacterial suf operon were identified in Arabidopsis, all of which appear to be plastid localized. AtSufC/AtNAP7 is a plastidic ABC/ATPase essential for Arabidopsis embryogenesis, which can rescue SufC deficiency in E. coli (Xu and Møller, 2004). AtSufC/AtNAP7 interacts with AtSufD/AtNAP6 and AtSufB/AtNAP1 in plastids (Xu et al, 2005), and this SufBCD complex may act as an energizer during iron acquisition. AtSufD/AtNAP6 also plays an essential role during Arabidopsis embryogenesis (Hjorth et al, 2005). Furthermore, the SufA-like protein CpIscA may act as a plastidic scaffold protein during Fe–S cluster assembly (Abdel-Ghany et al, 2005). Although the Arabidopsis genome harbors a SufE homolog, no data exist on this protein.

In chloroplasts Fe–S clusters are paramount for the functioning of cytochrome b/f complex, ferredoxin and photosystem I, ensuring thylakoid electron transport (Kapazoglou et al, 2000). The chloroplast import protein Tic55 also contains an Fe–S cluster (Caliebe et al, 1997). The requirement for Fe–S proteins to be part of multiple cellular processes argues that Fe–S cluster biogenesis is an essential part of plant development. Although recent studies have shed light on SUF-mediated iron acquisition in plastids, little is known regarding sulfur mobilization. Similarly, the intercompartmental coordination of plastidic and mitochondrial Fe–S cluster systems remains unexplored. Here we show that AtSufE in Arabidopsis represents an evolutionarily conserved SufE protein that, in contrast to other SUF proteins, localizes to both plastids and mitochondria, where it interacts with and activates the plastidic AtSufS and mitochondrial AtNifS1 desulfurases. AtSufE-mediated desulfurization activation is essential in both organelles, and we propose that AtSufE may act as an interorganellar coordinator of Fe–S cluster biogenesis in plants.

Results

AtSufE is an evolutionarily conserved SufE protein

A full-length Arabidopsis thaliana cDNA (1116 nt) encoding a putative SufE-like protein was cloned, which we named AtSufE. AtSufE is a single-copy nuclear gene (At4g26500) on chromosome IV encoding a 371-amino-acid protein (NM_118783) with 49% similarity to SufE from Nostoc punctiforme (NP_487553) and 27% similarity to E. coli SufE (NP_416194) (Figure 1B). In constrast to E. coli and N. punctiforme SufE, AtSufE contains a 150-amino-acid C-terminal extension, 88 amino acids of which show 41% similarity to the E. coli BolA protein (NP_487553) (Figure 1B). In E. coli, BolA acts as a morphogen, with overexpression resulting in spherical cells during starvation (Aldea et al, 1988); however, overexpression of the AtSufE BolA domain in isolation has no effect in E. coli or Arabidopsis (data not shown), indicating that the AtSufE BolA domain is a nonfunctional evolutionary relic.

Figure 1.

Figure 1

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AtSufE is a SufE homolog. (A) The AtSufE domain structure showing the presence of the transit peptide, the SufE and BolA domain. (B) The N-terminal region of AtSufE shows similarity to SufE proteins from E. coli (E. coli SufE) and N. punctiforme (Nostoc SufE), while the 88-amino-acid C-terminal region shows similarity to E. coli BolA (E. coli BolA). The SufE and BolA domains are separated by a 62-amino-acid linker region. AtSufE contains the conserved cysteine residue (amino acid 128, asterisk) found in other SufE proteins. The open arrowhead indicates the mitochondrial transit peptide and the filled arrowhead indicates the full-length transit peptide.

To test whether AtSufE represents an evolutionarily conserved SufE protein, we analyzed whether AtSufE could complement SufE-deficient E. coli during iron starvation. We generated an E. coli SufE mutant (MG1655ΔSufE) and compared its growth characteristics to wild type (WT) (MG1655) in the presence of the iron chelator 2,2′dipyridyl. MG1655ΔSufE is unable to grow in the absence of iron, while MG1655 shows no growth differences (Figure 2A). We then analyzed the effect of AtSufE expression in MG1655ΔSufE cells (MG1655ΔSufE AtSufE) in the absence of iron, revealing that AtSufE can complement the growth defects in SufE-deficient E. coli (Figure 2A).

Figure 2.

Figure 2

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AtSufE is evolutionarily conserved. (A) Complementation of SufE-deficient E. coli with AtSufE during iron starvation. WT E. coli (MG1655), SufE-deficient E. coli (MG1655ΔSufE) and MG1655ΔSufE expressing AtSufE (MG1655ΔSufE AtSufE) were plated on LB media and on LB media containing 200 μM 2,2′dipyridyl. On LB media all strains grew equally well. On LB media lacking iron MG1655ΔSufE showed no growth, while expression of AtSufE in MG1655ΔSufE restored growth, demonstrating complementation. (B) Complementation of MG1655ΔSufE by the SufE domain. MG1655, MG1655ΔSufE and MG1655ΔSufE expressing the AtSufE SufE domain (MG1655ΔSufE AtSufE-E) were grown in minimal A media with 2,2′dipyridyl, showing that the AtSufE SufE domain has retained its activity.

To test whether the AtSufE SufE domain is functional, we expressed the 220-amino-acid SufE domain (Figure 1B) in MG1655ΔSufE. This strain (MG1655ΔSufE AtSufE-E) was grown alongside MG1655 and MG1655ΔSufE cells in minimal A media containing 2,2′dipyridyl. MG1655ΔSufE shows growth retardation compared to MG1655, while MG1655ΔSufE AtSufE-E cells show partial complementation (Figure 2B). Combined, these data demonstrate that AtSufE is an evolutionarily conserved SufE protein.

AtSufE localizes to both plastids and mitochondria

AtSufE contains a 66-amino-acid N-terminal extension predicted to be a plastid-targeting transit peptide (P-TP) (Figure 1B). However, further predictions revealed a 30-amino-acid mitochondrial-targeting transit peptide, suggesting dual targeting (Figure 1B). To test this, constructs containing the full-length AtSufE cDNA fused to YFP were transiently expressed in tobacco and Arabidopsis, and analysis revealed fluorescence in both chloroplasts and mitochondria, demonstrating dual targeting (Figure 3A). To verify that both the plastid- and mitochondrial-targeting signals were present in the N-terminal extension, a truncated version of AtSufE lacking the 66-amino-acid N-terminus (AtSufEtrun) was fused to YFP, showing cytosolic localization (Figure 3B). To validate the observed cellular structures, the 65-amino-acid P-TP of AtSufC/AtNAP7 (Xu and Møller, 2004) and the 68-amino-acid mitochondrial-targeting transit peptide (M-TP) of the mitochondrial β subunit ATP synthase (Boutry et al, 1987) were fused to YFP. Transient expression analysis verified that the cellular structures showing AtSufE localization are indeed chloroplasts (Figure 3C) and mitochondria (Figure 3D). These results demonstrate that AtSufE is localized to both plastids and mitochondria.

Figure 3.

Figure 3

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AtSufE localizes to both plastid and mitochondria. All constructs were transiently expressed in tobacco leaves and YFP fluorescence is shown. (A) Full-length AtSufE-YFP localizes to chloroplasts (Chl) and mitochondria (Mito). (B) Removal of the 66-amino-acid transit peptide results in cytosolic distribution. The nucleus is indicated. (C) Expression of the 65-amino-acid AtSufC plastid-targeting transit peptide fused to YFP, demonstrating chloroplast structures. (D) Expression of the 68-amino-acid mitochondrial β subunit ATP synthase signal peptide fused to YFP, demonstrating mitochondrial structures. Scale bar=10 μm.

AtSufE is essential for normal embryo development

To analyze the function of AtSufE in Arabidopsis, we identified two SALK (The Salk Institute, USA) T-DNA insertion lines N800113 and N511580 (Alonso et al, 2003), containing T-DNA insertions in AtSufE (Figure 4). To verify the T-DNA insertion sites, AtSufE- and T-DNA-specific primers (Figure 4A) were used to PCR amplify the flanking regions (Figure 4B). N800113 has two adjacent T-DNAs at nucleotide positions 450 and 479, while N511580 contains one T-DNA at nucleotide position 657 (Figure 4A).

Figure 4.

Figure 4

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AtSufE deficiency results in abnormal seed development and arrested embryo development. (A) Schematic diagram showing the T-DNA insertion sites in N800113 and N511580 and primers used for molecular analysis. (B) PCR analysis of heterozygous N511580 using primers LBb1, RP1580 and LP1580, showing the presence of the T-DNA in N511580, its absence in WT and the presence of WT AtSufE (genomic) in both backgrounds. (C) WT siliques (a) showing uniform seed development and heterozygous N511580 (b), and N800113 (c) siliques showing ∼25% aborted seed (white arrows). (D) DIC microscopy of WT and homozygous aborted seeds from the same heterozygous N511580 siliques at (a, b) the two–four-cell stage, (c, d) the eight-cell stage and (e, f) the globular stage. Ep, embryo proper; Su, suspensor; Ac, apical cell; Bc, basal cell; Qc, prospective quiescent center; Co, prospective columella. (E) Transmission electron microscopy of WT (a) and N511580 (b) preglobular embryo cells with normal undifferentiated plastids (Pl) and mitochondria (Mi). N511580 (b) contains large vacuoles (V). Nu, nucleus. (F) Expression of a CaMV35S-AtSufE transgene in heterozygous N511580 plants, demonstrating successful complementation. WT (a), complemented N511580 (b) and a mutant embryo (c) from a silique with heart-stage WT embryos are shown. Ep, embryo proper; Su, suspensor. Successful complementation was verified by determining the ratio of viable to aborted seeds, showing a 15:1 ratio. Scale bars=20 μm in (D) and (F); 5 μm in (E).

We investigated seed structures in developing seed pods (siliques) in segregating populations of N800113 and N511580 plants. Siliques from heterozygous T3 populations were dissected, showing that, in contrast to uniformly developing green seeds in WT (Figure 4C), heterozygous atsufE siliques contained aborted seeds (Figure 4C). This phenotype was identical in N800113 and N511580 siliques (Figure 4C), showing a 3:1 ratio of viable to nonviable seeds. This is consistent with a recessive lethal segregation of embryos homozygous for the AtSufE T-DNA.

WT and homozygous N511580 embryos were further compared at the same developmental stages (same siliques). In general, N511580 embryos showed retarded development (Figure 4D): At the WT two–four-cell stage N511580 embryos had only reached initial zygote division, and at the WT eight-cell stage N511580 embryos had only reached the four-cell stage (Figure 4D). N511580 embryos never progressed beyond the preglobular eight-cell stage (Figure 4D). To analyze the ultrastructure of WT and N511580 preglobular embryos, we performed electron microscopy. N511580 embryo cells were more vacuolated than WT; however, the morphology and number of plastids and mitochondria were similar in WT and N511580 (Figure 4E), indicating that AtSufE loss of function does not disrupt overall organelle morphology as in AtSufC-deficient embryos (Xu and Møller, 2004).

Although N800113 and N511580 mutants showed identical phenotypes, we transformed a CaMV35S-AtSufE WT transgene into heterozygous N511580 plants for complementation. Seven out of 10 transgenic plants analyzed showed embryo development restoration and a 15:1 ratio of viable to nonviable seeds (Figure 4F), demonstrating that the embryo lethality is due to AtSufE deficiency.

AtSufE activity restoration in plastids or mitochondria is not sufficient to re-establish embryo development

The dual targeting of AtSufE to both plastids and mitochondria is unique to the SUF system in that all other SUF proteins in Arabidopsis are exclusively plastid localized (Møller et al, 2001; Pilon-Smits et al, 2002; Xu and Møller, 2004; Abdel-Ghany et al, 2005). To analyze whether AtSufE targeting to both organelles is important for embryo development, we tested if AtSufE activity restoration in either organelle could rescue the embryo lethality in AtSufE loss-of-function mutants. We generated CaMV35S binary vectors containing AtSufEtrun, lacking the endogenous transit peptide (Figure 3B), fused to either the AtSufC/AtNAP7 (Xu and Møller, 2004) 65-amino-acid P-TP or to the 68-amino-acid mitochondrial-targeting transit peptide (M-TP) of the mitochondrial β subunit ATP synthase (Boutry et al, 1987) generating P-TP/AtSufE and M-TP/AtSufE. To verify correct targeting, P-TP/AtSufE and M-TP/AtSufE were fused to YFP and transiently expressed in tobacco leaves, showing exclusive plastid (Figure 5B) and mitochondrial (Figure 5C) localization, respectively. The P-TP/AtSufE and M-TP/AtSufE binary vectors lacking YFP, together with the CaMV35S full-length WT AtSufE binary vector as a control, were then transformed into heterozygous N511580 plants and analyzed for restoration of embryo development. Five transgenic lines from each transformation event were analyzed by RT–PCR, showing the presence of AtSufE (Figure 5A), P-TP/AtSufE (Figure 5B) or M-TP/AtSufE transcripts (Figure 5C). Seed phenotypes were then analyzed from all 15 transgenic plants, revealing that, in contrast to N511580 plants transformed with AtSufE showing embryo development restoration (15:1 ratio; Figure 5A), mutants transformed with either P-TP/AtSufE (Figure 5B) or M-TP/AtSufE (Figure 5C) showed a seed phenotype (ratio 3:1) similar to the AtSufE loss-of-function mutants. Further analysis revealed that embryos in P-TP/AtSufE or M-TP/AtSufE plants were unable to progress beyond the preglobular stage (data not shown).

Figure 5.

Figure 5

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Normal embryo development requires AtSufE activity in both plastids and mitochondria. (A) WT TP-AtSufE-YFP, (B) P-TP/AtSufE-YFP, and (C) M-TP/AtSufE-YFP showing correct organelle targeting in tobacco. WT TP-AtSufE, P-TP/AtSufE and M-TP/AtSufE binary vectors were transformed into heterozygous N511580 and analyzed by RT–PCR, showing the presence of (A) WT TP-AtSufE, (B) P-TP/AtSufE and (C) M-TP/AtSufE transcripts. Seed phenotypes were analyzed, demonstrating that, in contrast to N511580 transformed with WT AtSufE (A; 15:1 ratio), N511580 transformed with either P-TP/AtSufE (B) or M-TP/AtSufE (C) showed a seed phenotype (ratio 3:1) similar to N511580. (D) Successful complementation of N511580 by both the P-TP/AtSufE and M-TP/AtSufE transgenes. RT–PCR analysis of homozygous N511580 plants (A, B), revealing the absence of WT TP-AtSufE but the presence of both P-TP/AtSufE and M-TP/AtSufE transcripts. WT and complemented viable seedlings are shown.

As a control to ensure that both P-TP/AtSufE and M-TP/AtSufE are functional proteins in their respective organelles, we assayed whether the two separate transgenes could rescue the homozygous N511580 mutant lethal phenotype. Heterozygous plants containing either the P-TP/AtSufE or the M-TP/AtSufE transgenes were crossed by pollination and the resulting seed screened for viability. In all, 20 viable seedlings were analyzed by RT–PCR, revealing that two seedlings (Figure 5D; seedlings A and B) did not contain the WT AtSufE transcript but showed the presence of P-TP/AtSufE and M-TP/AtSufE transcripts (Figure 5D). The fact that the two independent transgenes can fully complement the homozygous N511580 mutant (Figure 5D) demonstrates that both the plastid-localized and the mitochondria-localized AtSufE proteins used in this experiment are functional in their respective organelles.

AtSufE interacts with AtSufS and AtNifS1 in vivo

To test whether AtSufE could interact with the plastidic cysteine desulfurase AtSufS (Leon et al, 2002; Pilon-Smits et al, 2002; Ye et al, 2005) and the putative mitochondrial cysteine desulfurase AtNifS1 (Kushnir et al, 2001), we performed yeast two-hybrid assays. Full-length AtSufE, AtSufS and AtNifS1 proteins fused to the GAL4 activation domain (Figure 6A; AD-AtSufE, AD-AtSufS, AD-AtNifS1) and to the GAL4 DNA-binding domain (Figure 6A; BD-AtSufE, BD-AtSufS, BD-AtNifS1) were expressed in HF7c yeast cells and monitored for growth in the absence of histidine (His) as a marker for protein–protein interaction. His auxotrophy was restored in cells coexpressing AD-AtSufE/BD-AtSufS and AD-SufS/BD-AtSufE, demonstrating that AtSufE interacts with AtSufS (Figure 6A). Furthermore, cells coexpressing AD-AtSufE/BD-AtNifS1 and AD-AtNifS1/BD-AtSufE showed growth on media lacking His (−HTL), revealing that AtSufE also interact with AtNifS1 (Figure 6A). Yeast cells coexpressing the AD or BD vectors with BD-AtSufE, BD-AtSufS, BD-AtNifS1 or AD-AtSufE, AD-AtSufS, AD-AtNifS1, respectively, showed no growth on -HTL media (Figure 6A). To gain insight into the relative interaction strengths, the ratio of yeast growth on media lacking tryptophan and leucine (-TL) to growth on -HTL media was determined, revealing that the AtSufE/AtSufS interaction is stronger than the AtSufE/AtNifS1 interaction.

Figure 6.

Figure 6

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AtSufE interacts with plastidic AtSufS and mitochondrial AtNifS1 in vivo. (A) Yeast two-hybrid analysis of AtSufE, AtSufS and AtNifS1. Full-length AtSufE, AtSufS and AtNifS1, fused to the GAL4 activation domain (AD-AtSufE, AD-AtSufS, AD-AtNifS1) and to the GAL4 DNA-binding domain (BD-AtSufE, BD-AtSufS, BD-AtNifS1), were expressed in HF7c yeast cells and monitored for the ability to grow without histidine (-HTL media) after 3 days. Histidine auxotrophy was restored in cells coexpressing AD-AtSufE/BD-AtSufS, AD-AtSufS/BD-AtSufE, AD-AtSufE/BD-AtNifS1 and AD-AtNifS1/BD-AtSufE, showing that AtSufE interacts with AtSufS and AtNifS1 in yeast. Yeast coexpressing the above vectors with empty AD or BD vectors showed no growth on -HTL media. Relative interaction strengths were determined by measuring the ratio of growth on media lacking tryptophan and leucine (-TL) to growth on -HTL media. (B) BiFC assays. A nonfluorescent N-YFP fused to the C-terminus of AtSufE was transiently coexpressed in tobacco leaf cells with either AtSufS or AtNifS1 fused to the N-terminus of a nonfluorescent C-YFP. In cells expressing AtSufE-N-YFP and AtSufS-C-YFP YFP fluorescence occurs in chloroplasts, while in cells expressing AtSufE-N-YFP and AtNifS1-C-YFP fluorescence occurs in mitochondria.

To confirm the observed protein interactions in living plant cells, we performed bimolecular fluorescence complementation (BiFC) assays (Hu et al, 2002) based on the reconstitution of YFP fluorescence when nonfluorescent N-terminal (N-YFP) and C-terminal (C-YFP) YFP fragments are brought together by two interacting proteins. AtSufE fused to the N-terminus of N-YFP and AtSufS and AtNifS1 fused to the N-terminus of C-YFP were expressed in tobacco leaves. Cells coexpressing AtSufE-N-YFP and AtSufS-C-YFP showed YFP fluorescence in chloroplasts, while cells coexpressing AtSufE-N-YFP and AtNifS1-C-YFP showed YFP fluorescence in mitochondria (Figure 6B), demonstrating that AtSufE interacts with AtSufS and AtNifS1 in planta. To ensure that the restored fluorescence was not due to nonspecific interactions, we coexpressed AtSufE-N-YFP with the plastid-localized AtNAP7-C-YFP (Xu and Møller, 2004), two proteins that show no interaction in yeast cells, observing no fluorescence (data not shown).

AtSufS- and AtNifS1-mediated cysteine desulfurization is activated by AtSufE

The interaction of AtSufE with AtSufS and AtNifS1 prompted us to analyze whether AtSufE could stimulate cysteine desulfurization. We expressed and purified 6xHis-AtSufE, 6xHis-AtSufS and 6xHis-AtNifS1 proteins (Figure 7A) and performed in vitro cysteine desulfurization assays. In the absence of AtSufE, AtNifS activity was almost undetectable; however, in the presence of AtSufE cysteine desulfurization increased ∼30-fold (Figure 7B). This demonstrates not only that AtNifS1 is a cysteine desulfurase but also that activity is dependent on AtSufE activation. In constrast, AtSufS showed basal cysteine desulfurase activity in the absence of AtSufE, with an ∼7-fold increase in activity in the presence of AtSufE (Figure 7B), suggesting that AtSufE is not strictly essential for AtSufS-mediated desulfurization.

Figure 7.

Figure 7

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AtSufE acts as an activator of AtSufS- and AtNifS1-mediated cysteine desulfurization. (A) SDS–PAGE of purified 6xHis-AtSufE, 6xHis-AtSufS and 6xHis-AtNifS1 proteins. (B) Desulfurase assays using 200 nM 6xHis-AtSufS, 6xHis-AtNifS1 and 6xHis-atSufE. Methylene blue formation was measured at 670 nm. Na2S (1–100 μM) was used for calibration and sulfur release is shown.

Elevated levels of AtSufE result in chlorosis and retarded plant development

To further analyze the in vivo role of AtSufE during Fe–S cluster biogenesis, we generated transgenic plants containing a CaMV35S-AtSufE transgene (Figure 8A). Four transgenic lines showing AtSufE overexpression (AtSufE-overexpressing (AtSufE-ox)) were analyzed, showing growth retardation and chlorosis, particularly in younger leaves and siliques (Figure 8B). As AtSufE enhances AtSufS and AtNifS1 desulfurization activity (Figure 7B), the observed phenotype suggests enhanced desulfurization, resulting in increased cellular sulfide levels. To test this, we performed in vivo cysteine desulfurase activity assays on protein extracts from WT and AtSufE-ox plants and found significantly elevated sulfur levels in response to AtSufE overexpression (Figure 8C). To further examine whether this in vivo increase in cysteine desulfurization activity had an affect on Fe–S cluster-containing proteins, we carried out Western blot analysis using antibodies raised against the Rieske protein and PsaC, both plastidic Fe–S proteins. In the absence of functional Fe–S cluster biosynthesis many Fe–S proteins are unstable, and in line with this we found that elevated AtSufE-mediated cysteine desulfurase activity resulted in increased levels of both Rieske and PsaC protein (Figure 8D). This suggests that increased flux through the sulfur branch of the SUF Fe–S cluster biogenesis pathway increases Fe–S cluster formation, ultimately leading to higher stability of Fe–S proteins.

Figure 8.

Figure 8

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Overexpression of AtSufE in transgenic plants leads to retarded growth and chlorosis. (A) A CaMV35S-AtSufE binary vector was transformed into WT Arabidopsis. Four transgenic lines with increased AtSufE transcript (AtSufE-ox, A–D) compared to WT (A, B) were selected for analysis. Actin was used a control. (B) WT and AtSufE-ox plants were grown for 2 weeks on soil, revealing that AtSufE-ox plants show chlorosis and retarded growth in leaves and siliques. (C) In vivo cysteine desulfurization analysis showing that AtSufE-ox plants contain higher levels of cellular sulfur. (D) Western blot analysis demonstrating that the plastidic Fe–S proteins Rieske and PsaC are more abundant in AtSufE-ox plants compared to WT. (E) RT–PCR analysis of WT and AtSufE-ox plants showing that AtSufE overexpression results in AtNifS1 and AtSufS transcript induction, while AtSufB, AtSufD and AtSufC transcript levels remain unchanged. Actin was used as control.

As AtSufE stimulates AtSufS and AtNifS1 activities, we tested whether AtSufE overexpression affects AtSufS and AtNifS1 levels. RT–PCR revealed that both transcripts are induced (Figure 8E), implying that increased cysteine desulfurization in AtSufE-ox plants is due to elevated levels of the AtSufE/AtSufS and AtSufE/AtNifS1 complexes. In contrast, AtSufB, AtSufC and AtSufD transcripts are not affected in AtSufE-ox plants (Figure 8C), suggesting that the sulfur mobilization and iron acquisition branches of the SUF protein-mediated Fe–S cluster biogenesis pathways may show independent regulation.

Discussion

SUF protein-mediated Fe–S cluster biogenesis in plastids can be divided into two main processes: iron acquisition and sulfur mobilization from cysteine. Recent studies have shed light on iron acquisition (Xu and Møller, 2004; Hjorth et al, 2005; Xu et al, 2005), but little is known about how the SUF system regulates sulfur mobilization. We have characterized AtSufE, revealing dual localization to plastids and mitochondria. AtSufE interacts with and activates the cysteine desulfurases, AtSufS in plastids and AtNifS1 in mitochondria, and both activations are vital during embryogenesis. Our data demonstrate that plastidic and mitochondrial Fe–S cluster assemblies share a common, vital component, and we propose that AtSufE acts as an interorganellar coordinator of Fe–S cluster biogenesis in Arabidopsis.

AtSufE is an evolutionarily conserved SufE protein in Arabidopsis with dual localization

As E. coli SufE mobilizes sulfur preferably under iron-limiting conditions (Outten et al, 2004), the finding that AtSufE can complement SufE-deficient E. coli in the absence of iron indicates the evolutionary conservation of the process (Figure 2A). Moreover, that the SufE domain is sufficient for complementation suggests that this domain has retained its prokaryotic SufE activity (Figure 2B). E. coli SufE acts as a sulfur acceptor, mobilized by SufS, where cysteine 51 is the acceptor site (Figure 1B; Outten et al, 2003) and AtSufE contains this conserved cysteine (Figure 1B). As AtSufE can substitute its bacterial counterpart, AtSufE-mediated sulfur mobilization probably shows similarities to that in bacteria.

Despite previous studies suggesting that the entire SUF pathway is exclusively plastid localized (Balk and Lobreaux, 2005), AtSufE is unique in that it is targeted to both plastids and mitochondria (Figure 3A). Although it is not known whether amino acids responsible for plastid and mitochondrial targeting are overlapping or separate (Chew et al, 2003), deletion experiments show that signals for the dual localization lie within the 66-amino-acid N-terminal extension of AtSufE (Figure 3B). Dual SUF protein localization suggests that Fe–S cluster biogenesis may be controlled by a common protein acting in both organelles.

Fe–S cluster biogenesis is an essential process in both plastids and mitochondria during embryogenesis

The embryo lethal phenotype of homozygous AtSufE loss-of-function mutants demonstrates that AtSufE is an essential protein (Figure 4D). Through yeast two-hybrid and BiFC analysis, we have shown that AtSufE interacts with AtSufS in plastids (Figure 6) and AtNifS1 in mitochondria (Figure 6), suggesting that AtSufE acts as the essential protein in the AtSufE/AtSufS and AtSufE/AtNifS1 complexes. AtSufS or AtNifS1 loss-of-function analysis has not been reported in plants; however, in Synechocystis sp. PCC6803 deletion of the slr0077/SufS gene results in only merodiploid strains (Seidler et al, 2001), while in Bacillus subtilis the SufS homolog csd/yurw is essential for viability (Kobayashi et al, 2003). This suggests that AtSufE, AtSufS and AtNifS1 may all be essential during sulfur mobilization.

The lethality in AtSufE loss-of-function embryos is more severe than that observed in AtSufC/AtNAP7 mutants. Embryo development in atsufE is severely retarded and arrests at the preglobular stage (Figure 4D), while atsufC embryos arrest at the late globular stage (Xu and Møller, 2004). The severity of the atsufE phenotype may simply be due to the combined loss of sulfur mobilization in both plastids and mitochondria. We suggest that AtSufE-mediated sulfur mobilization may represent a more universal step involved in different Fe–S cluster assembly pathways. To test this notion, we restored AtSufE activity specifically in plastids or mitochondria (Figure 5). Interestingly, plants harboring only plastid-targeted or only mitochondrial-targeted AtSufE (Figure 5B and C) showed no embryo development restoration (Figure 5), demonstrating that organelle-specific AtSufE re-establishment is not sufficient to allow embryo progression, revealing that AtSufE-mediated sulfur mobilization is crucial in both organelles.

Plastidic Fe–S cluster biogenesis is mediated by the SUF proteins and in mitochondria the presence of AtNifS1 and all key ISC components (cf. Balk and Lobreaux, 2005) suggests the presence of at least two pathways in both organelles. That AtSufE deficiency in either organelle results in embryo lethality (Figure 5) implies little functional redundancy between the SUF and ISC systems. This supports the notion that plants have multiple Fe–S cluster biogenesis pathways, separated in two organelles, which to date have been largely viewed as independent processes.

Activation of AtSufS and AtNifS1 cysteine desulfurization by AtSufE may coordinate Fe–S cluster biogenesis

The finding that AtSufE interacts with and activates AtSufS and AtNifS1 demonstrates that the AtSufE/AtSufS and AtSufE/AtNifS1 complexes are functional (Figure 7B). Furthermore, an increase in AtSufE results in AtSufS and AtNifS1 induction (Figure 8E), suggesting a universal regulation of proteins involved in sulfur mobilization. Furthermore, it appears that correct AtSufE levels are vital in that AtSufE overexpression leads to growth retardation and chlorosis due to increased sulfur levels (Figure 8B and C).

AtNifS1 and AtSufS differ in their cysteine desulfurization activities. In the absence of AtSufE AtNifS1-mediated desulfurization is almost undetectable, while AtSufS shows significant activity in the absence of AtSufE (Figure 7B). This suggests that mitochondrial AtNifS1-mediated desulfurization is dependent on AtSufE, while plastidic AtSufS activity is not dependent, but stimulated by AtSufE. The stimulation by AtSufE in plastids is, however, essential because restoration of AtSufE in mitochondria does not result in embryo development restoration (Figure 5C). This implies that the low basal level of sulfur mobilization by AtSufS, in the absence of AtSufE, is not sufficient to support adequate Fe–S cluster biogenesis, ensuring plastid functionality.

In E. coli, transfer of sulfur to SufE occurs in the presence of SufS but not NifS (Outten et al, 2003); so why have plants evolved a common activator of sulfur mobilization in two different organelles? Plant Fe–S cluster biogenesis clearly takes place in both plastids and mitochondria (Balk and Lobreaux, 2005), and we have shown that not only is the presence of sulfur mobilization in both organelles vital but also that appropriate rates of cysteine desulfurization appear important (Figures 5 and 8). Therefore, plant cells probably require communication between plastids and mitochondria to ensure coordination and appropriate Fe–S cluster assembly rates. We propose that AtSufE may fulfil this role, acting as an interorganellar coordinator ensuring a balance between plastidic and mitochondrial Fe–S cluster biogenesis (Figure 9). As plastidic and mitochondrial Fe–S protein biogenesis varies during development, it is possible that the regulation of AtSufE-mediated cysteine desulfurization is influenced by developmental and environmental cues (Figure 9). By exploring in vivo Fe–S cluster synthesis rates in plastids and mitochondria, with respect to AtSufE activity, under different growth conditions and at different developmental stages, the complexity of Fe–S cluster biogenesis coordination in plants will become unraveled.

Figure 9.

Figure 9

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A working model of AtSufE-mediated coordination of Fe–S cluster biogenesis in plastids and mitochondria in Arabidopsis. AtSufE (E) is targeted to both plastids and mitochondria, where it interacts with plastidic AtSufS (S) and mitochondrial AtNifS1 (S1). This interaction results in activation of cysteine desulfurization by AtSufS and AtNifS1, resulting in sulfur transfer to the acquired iron to generate Fe–S clusters. The targeting of AtSufE (E) to both plastids and mitochondria may represent a control point possibly influenced by endogenous developmental cues or exogenous environmental cues.

Materials and methods

Isolation of AtSufE T-DNA insertion mutants and complementation

Plants were grown on MS medium or soil under 16 h light/8 h dark cycles at 22°C. N511580 and N800113 were identified from the SALK T-DNA collection (Alonso et al, 2003) and genotyped by PCR using primers LP1580/LB1 (N511580) and LP1580/LB3 or RP1580/LB3 (N800113). (Primers are listed in Supplementary Table.) For complementation analysis, full-length AtSufE was PCR amplified with primers ATSUFE-BA-L/ATSUFE-BA-R, digested with XhoI and SpeI, cloned into the CaMV35S promoter binary vector pBA002 (Kost et al, 1998), and transformed into heterozygous N511580 plants using the floral-dip method (Clough and Bent, 1998).

For organelle-specific complementation, truncated AtSufE, lacking its transit peptide, was PCR amplified with primers ATSUFE-Trun-L/ATSUFE-BA-R, digested with XhoI and SpeI, and ligated in pBA002, resulting in pBA-trun-AtSufE. The AtSufC/AtNAP7 transit peptide was PCR amplified using primers Chl-lead-L/Chl-lead-R and the ATP synthase mitochondrial signal peptide using Mito-lead-L/Mito-lead-R, digested with XhoI and ligated into pBA-trun-AtSufE. For localization studies, the AtSufE versions were PCR amplified from pBA-Chl-trun-atSufE and pBA-Mito-trun-atSufE using primer pairs Chl-lead-L/ATSUFE-W18-R and Mito-lead-L/ATSUFE-W18-R, cloned upstream of YFP in pWEN18 and transiently expressed in tobacco.

To ensure that pBA-Chl-trun-atSufE and pBA-Mito-trun-atSufE in combination could rescue the N511580 phenotype, heterozygous N511580 plants containing pBA-Chl-trun-atSufE or pBA-Mito-trun-atSufE were genotyped for the T-DNA insertion and for either the Chl-trun-atSufE or Mito-trun-atSufE transgenes as before. Positive plants were then crossed and the seed germinated on MS media containing Basta (10 mg/l). PCR was performed using primers LBb1 with LP5180 and RP5180 to screen for homozygous seedlings. RNA was then extracted from the homozygous seedlings, followed by RT–PCR analysis using primers specific for WT AtSufE, Chl-trun-atSufE and Mito-trun-atSufE as described above.

Overexpression and purification of AtSufE, AtSufS and AtNifS1

Full-length AtSufE, AtSufS and AtNifS1 cDNAs were PCR amplified using primers AtSufE-ET-L/AtSufE-ET-L, AtSufS-ET-L/AtSufS-ET-R and AtNifS-ET-L/AtNifS-ET-R, AtSufE digested with EcoRI and XhoI, AtSufS and AtNifS with BamHI and XhoI, and ligated into pET28a (Novagen). The plasmids were transformed into E. coli Rosetta(DE3)pLysS (Novagen) and expression performed for 20 h in auto-induction ZYM-5052 media (Studier, 2005). Cells were disrupted in 50 mM Tris–HCl (pH 8.0), 25% sucrose, 5 mM MgCl2, 100 mM NaCl, 1% Triton X-100 and 10 μM/ml Benzonase® Nuclease (Novagen), proteins purified using TALON affinity resin (BD Biosciences) and protein purity verified by SDS–PAGE.

Cysteine desulfurase assay

The procedure followed previous protocols (Outten et al, 2003) with modifications. Reactions were carried out in 50 mM Tris–HCl, pH 7.5, 5 mM MgCl2 and 100 mM NaCl using 200 nM purified 6xHis-AtSufS, 6xHis-AtNifS1 and 6xHis-AtSufE. BSA (200 nM) was used as a control. Pyridoxal 5′-phosphate (10 μM) and DTT (100 μM) were used for all reactions. Reactions were initiated by adding 100 μM L-cysteine, allowed to proceed for 30 min at 30°C, and were quenched by adding 100 μl of 20 mM N,N-dimethyl-p-phenylenediamine/7.2 M HCl. The addition of 100 μl of 30 mM FeCl3/1.2 M HCl followed by incubation for 20 min led to formation of methylene blue, which was measured at 670 nm. Na2S (1–100 μM) was used for calibration.

For in vivo assays, WT and AtSufE-ox seedlings were grown for 2 weeks and proteins extracted as described (Pilon-smits et al, 2002): 2 g fresh tissue was added to 2 ml extraction buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM phenylmethyl sulfonyl fluoride, 1 mM DTT, 0.5% (v/v) Triton X-100) and 100 μl of each protein solution was used for enzyme assays as described.

Western blotting

WT and two independent AtSufE-ox seedlings were grown for 2 weeks, total protein extracted, followed by Western blot analysis using antibodies raised in rabbit against the Rieske and PsaC proteins.

AtSufE localization analysis

Full-length AtSufE was PCR amplified using primers ATSUFE-W18-L/ATSUE-W18-R, digested with XhoI and KpnI, cloned into pWEN18 as an N-YFP and transiently expressed in tobacco, followed by YFP fluorescence analysis on a Nikon TE-2000U inverted microscope (Nikon, Japan). As a control for targeting, the mitochondrial and plastid signal peptides were PCR amplified as before and fused to YFP in pWEN18, followed by transient expression in tobacco.

For embryo analysis, seeds were cleared for 24 h in chloral hydrate/H2O/glycerol, 8:2:1 (w/v/v), and examined using DIC microscopy.

Electron microscopy was performed using standard protocols on a JEOL 1220 transmission electron microscope.

Generation of an E. coli SufE mutant and complementation by AtSufE

The SufE gene in E. coli strain MG1655 was disrupted using standard protocols (Datsenko and Wanner, 2000). AtSufE was PCR amplified using primers ATSUFE-UC-L/ATSUFE-UC-R, digested with PstI and KpnI and ligated in pUC19 to generate pUC-AtSufE. pUC-AtSufE-E, containing only the SufE domain of AtSufE, was generated by PCR amplification using primers ATSUFE-E-L/ATSUFE-E-R, digested with PstI and KpnI, and ligated in pUC19. pUC-AtSufE and pUC-AtSufE-E were transformed into MG1655ΔsufE to generate MG1655ΔsufE AtSufE and MG1655ΔsufE AtSufE-E. WT MG1655, MG1655ΔsufE, and MG1655ΔsufE AtSufE and MG1655ΔsufE AtSufE-E were grown overnight in LB medium at 37°C and plated on LB medium or used to inoculate minimal A medium containing 0.2% gluconate, 1 μg/ml thiamine and 200 μM 2,2′dipyridyl. Growth was measured at OD600.

Yeast two-hybrid analysis

Full-length AtSufE, AtSufS and AtNifS1 cDNAs were PCR amplified using primers ATSUFE-YTH-L/ATSUFE-YTH-R, ATSUFS-YTH-L/ATSUFS-YTH-R and ATNIFS1-YTH-L/ATNIFS1-YTH-R, AtSufE and AtNifS1 digested with EcoRI and BamHI, AtSufS with NdeI and BamHI, and cloned into pGADT7 and pGBKT7. Plasmids and empty vector controls were transformed into HF7c yeast cells and tested for restoration of His auxotrophy (Matchmaker two-hybrid system, version 3, Clontech).

Bimolecular fluorescence complementation

Full-length AtSufE was PCR amplified using primers ATSUFE-W18-L/ATSUFE-W18-R, digested with XhoI and KpnI, and cloned into pWEN-N-YFP (containing amino acids 1–154 of YFP), generating pWEN-AtSufE-N-YFP. Full-length AtSufS and AtNifS1 were PCR amplified using primers ATSUFS-WEN-L/ATSUFS-WEN-R and ATNIFS1-NY-L/ATNIFS1-NY-R, digested with XhoI and KpnI, and cloned into pWEN-C-YFP separately. pWEN-AtSufE-N-YFP/pWEN-AtSufS-C-YFP and pWEN-AtSufE-N-YFP/pWEN-AtNifS1-C-YFP were transiently expressed in tobacco in separate experiments and analyzed for YFP fluorescence.

Phenotypic analysis of AtSufE-ox plants

Full-length AtSufE was amplified using primers ATSUFE-W18-L/ATSUFE-BA-R, digested with XhoI and SpeI, ligated in pBA002, followed by transformation into Arabidopsis using the floral-dip method (Clough and Bent, 1998). WT and AtSufE-ox seeds were germinated on MS medium containing Basta (10 mg/l), transferred to soil after 10 days and phenotypes recorded after 2 weeks. RT–PCR was performed to analyze expression of AtSufB, AtSufD, AtSufC, AtSufS and AtNifS1 in WT and AtSufE-ox plants using primers ATNAP1-L/ATNAP1-R, ATNAP6-L/ATNAP6R, ATNAP7-L/ATNAP7-R, ATSUFS-L/ATSUFS-R and ATNIFS1-L/ATNIFS1-R. Actin was used as a control (primer ACT8L/ACT8R).

Supplementary Material

Supplementary Table 1

7600968s1.pdf (16.5KB, pdf)

Acknowledgments

We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants, NASC for providing seeds, Natalie Allcock and Stefan Hyman for electron microscopy, Sarah Deacon and Mike McPherson for auto-induction protocols, and Trudie Allen for crossing. We thank Ralf Bernd Klösgen for the Rieske protein antibody and Henrik Scheller for the PsaC antibody. This work was supported by grants from the BBSRC (91/P16510 and BB/C00552X/1) and The Royal Society (574006.G503/23280/SM) to SGM. SGM is the recipient of an EMBO Young Investigator Award.

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Supplementary Materials

Supplementary Table 1

7600968s1.pdf (16.5KB, pdf)

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