# Iron-sulfur protein NFU2 is required for branched-chain amino acid synthesis in Arabidopsis roots

Brigitte Touraine, Florence Vignols, Jonathan Przybyla-Toscano, Till Ischebeck, Tiphaine Dhalleine, Hui-Chen Wu, Cyril Magno, Nathalie Berger, Jérémy Couturier, Christian C. Dubos, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Brigitte Touraine, Florence Vignols, Jonathan Przybyla-Toscano, Till Ischebeck, Tiphaine Dhalleine, et al.. Iron-sulfur protein NFU2 is required for branched-chain amino acid synthesis in Arabidopsis roots. Journal of Experimental Botany, 2019, 70 (6), pp.1875-1889. 10.1093/jxb/erz050. hal-02095655

HAL Id: hal-02095655

https://hal.science/hal-02095655v1

Submitted on 25 Aug 2025

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
The iron-sulfur protein NFU2 plays a predominant role in branched-chain amino acid synthesis in
Arabidopsis roots

Brigitte Touraine<fnref n="1" />, Florence Vignols<fnref n="1" />,‡, Jonathan Przybyla-Toscano<fnref n="2" />,‡, Till Ischebeck<fnref n="3" />, Tiphaine Dhalleine<fnref n="2" />, Hui-Chen Wu<fnref n="1" />,¶, Cyril Magno<fnref n="1" />, Nathalie Berger<fnref n="1" />, Jérémy Couturier<fnref n="2" />, Christian Dubos<fnref n="1" />, Ivo Feussner<fnref n="3" />, Stefano Caffarri<fnref n="4" />, Michel Havaux<fnref n="5" />, Nicolas Rouhier<fnref n="2" />,§, Frédéric Gaymard<fnref n="1" />,§

Gaymard Frédéric, Biochimie et Physiologie Moléculaire des Plantes, CNRS/INRA/Université Montpellier 2, SupAgro. Bat 7, 2 place Viala, 34060 Montpellier cedex 1, France, Phone: ++33 6 08 03 37 34, Email: frederic.gaymard@inra.fr

Rouhier Nicolas, Université de Lorraine, UMR1136 Interactions Arbres/Microorganismes, F-54500 Vandœuvre-lès-Nancy, France, Phone: ++33 3 72 74 51 57, Email: nicolas.rouhier@univ-lorraine.fr

**Keywords:** *Arabidopsis thaliana*, branched-chain amino acids, chloroplast, development, iron-sulfur protein, photosynthesis

<footnote n="1">¹ BPMP, CNRS, INRA, Montpellier SupAgro, Univ Montpellier, Montpellier, France.</footnote>

<footnote n="2">² Université de Lorraine, Inra, IAM, F-54000 Nancy, France.</footnote>

<footnote n="3">³ Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences and Göttingen Center for Molecular Biosciences (GZMB), University of Göttingen, 37077 Göttingen, Germany.</footnote>

<footnote n="4">⁴ Aix-Marseille Université, CEA Cadarache, CNRS UMR 7265, Laboratoire de Génétique et Biophysique des Plantes, 13009 Marseille, France.</footnote>

<footnote n="5">⁵ CEA Cadarache, CNRS UMR 7265, Aix-Marseille Université, Laboratoire d'Ecophysiologie Moléculaire des Plantes, 13108, Saint-Paul-lez-Durance, France.</footnote>

<footnote>‡ These authors have equally contributed to the work.</footnote>

<footnote>§ To whom correspondence should be addressed:</footnote>
**Highlight**

The primary root growth defect of *nfu2* plants is rescued by branched chain amino acids, which is consistent with an impaired maturation of the [Fe₂S₂] cluster of a dihydroxyacid dehydratase.

**Abstract**

Numerous proteins require a metallic cofactor for their function. In plastids, the maturation of iron-sulfur (Fe-S) proteins necessitates a complex assembly machinery. We focused on *Arabidopsis thaliana* NFU1, NFU2 and NFU3, which participate in the late maturation steps. According to the strong photosynthesis defects observed in *high chlorophyll fluorescence 101* (*hcf101*), *nfu2* and *nfu3* plants, we determined that NFU2 and NFU3, but not NFU1, act immediately upstream from HCF101 for the maturation of [Fe₄S₄]-containing photosystem I subunits. An additional function of NFU2 in the maturation of the [Fe₂S₂] cluster of a dihydroxyacid dehydratase was obvious from the accumulation of precursors of the branched-chain amino acid synthesis pathway in *nfu2* roots and the rescue of the primary root growth defect by supplying branched-chain amino acids. The absence of NFU3 in roots precluded any compensation. Overall, unlike their eukaryotic and prokaryotic counterparts, which are specific of [Fe₄S₄] proteins, NFU2 and NFU3 contribute to the maturation of both [Fe₂S₂] and [Fe₄S₄] proteins either as a relay in conjunction with other proteins such as HCF101 or by directly delivering Fe-S clusters to client proteins. Considering the low number of Fe-S cluster transfer proteins relative to final acceptors, additional targets probably await identification.
Introduction

Several metabolic pathways and cellular processes rely on metallic cofactors such as iron-
sulfur (Fe-S) clusters. For instance, Fe-S proteins are required for photosynthesis, respiration, sulfur
and nitrogen assimilation, co-enzyme synthesis, DNA repair and replication or ribosome biogenesis
(Balk and Schaedler, 2014; Couturier et al., 2013; Lill, 2009). These Fe-S clusters are formed by iron
and sulfur atoms and are generally coordinated in proteins via cysteinyl residues, the most common
clusters being the diamond [Fe₂S₂] and the cubane [Fe₄S₄] clusters. In eukaryotes and in particular in
plants, three machineries called SUF (sulfur mobilization), ISC (iron-sulfur cluster) and CIA
(cytosolic iron-sulfur cluster assembly) are required for the maturation of plastidial, mitochondrial and
cytosolic/nuclear Fe-S proteins, respectively (Lill and Muhlenhoff, 2008). The cytosolic machinery
depends on the mitochondrial ISC system but not on the SUF system for providing the required sulfur
atoms (Bernard et al., 2009; Van Hoewyk et al., 2007). An ABC transporter, named ATM3 in plants,
would transport a glutathione-derived molecule, possibly glutathione polysulfide, across the
mitochondrial membranes (Kushnir et al., 2001; Schaedler et al., 2014). In mitochondria and plastids,
the required sulfur atoms are extracted from cysteine by pyridoxal phosphate-dependent cysteine
desulfurases, NFS1 and NFS2 respectively (Couturier et al., 2013). The nature of the molecule(s)
donating iron for achieving the first step(s) of the de novo Fe-S cluster assembly on scaffold proteins
has yet to be firmly established for all machineries. In a second step, the preformed Fe-S cluster is
conveyed to final acceptor proteins with the help of maturation factors including chaperones, Fe-S
cluster transfer or carrier proteins, which assemble themselves transiently the Fe-S cluster and a few
additional targeting factors that likely serve for recruiting these acceptor proteins.

The SUF system found in plastids has been inherited from bacteria. In *Escherichia coli*, the SUF operon encodes six proteins (SufA, B, C, D, E and S), is mostly expressed in response to external constraints, and co-exists with the ISC housekeeping system (Roche *et al.*, 2013). In both plants and bacteria possessing the SUF system, the SufS/NFS2 is involved in sulfur mobilization from cysteine, the SufE protein serves as an activator and the SufB, C and D proteins form a scaffold complex (Fontecave *et al.*, 2005; Hu *et al.*, 2017; Van Hoewyk *et al.*, 2007; Wollers *et al.*, 2010). The exact role of SufA/SUFA1 is debated but it could act as a Fe-S cluster transfer protein (Abdel-Ghany *et al.*, 2005; Chahal *et al.*, 2009; Gupta *et al.*, 2009). Moreover, there are additional players including the glutaredoxin 4 (Grx4), BolA, the P-loop NTPase Mrp, Ygfz, ErpA and NfuA, all of these most likely serving for the late steps (Roche *et al.*, 2013). Most of them have orthologs in plants. The chloroplastic Grx4 orthologs, GRXS14 and GRXS16, are hypothesized to serve as transfer proteins due to their capacities to bind and transfer labile Fe-S clusters to acceptor proteins as shown *in vitro* but also to complement a yeast mutant for the mitochondrial Grx5 ortholog (Bandyopadhyay *et al.*, 2008a). These GRXs were found to form complexes with plastidial proteins containing a BOLA domain *e.g.*
BOLA1, BOLA4 and SUFE1 (Couturier *et al.*, 2014). The plastidial ortholog of Mrp is called HCF101 for HIGH CHLOROPHYLL FLUORESCENCE 101. In accordance with the capacity of the recombinant protein to assemble a [Fe₄S₄] cluster, knock-down *Arabidopsis thaliana* plants in *HCF101* gene fail to accumulate mature PSI, which contains three [Fe₄S₄] clusters, and have reduced levels of ferredoxin-thioredoxin reductase (FTR) (Lezhneva *et al.*, 2004; Schwenkert *et al.*, 2009; Stockel and Oelmuller, 2004). Knock-out plants are seedling-lethal pointing to the central role of HCF101 for PSI assembly. Among the four SufA/ErpA/IscA family members found in plants, the sole plastidial isoform should be SUFA1. There is one Ygfz ortholog in plastids named IBA57.2 but its implication was not established so far (Waller *et al.*, 2012). Finally, regarding NFU family, three plastid-located members, named NFU1, NFU2 and NFU3, have been identified in *A. thaliana* whereas NFU4 and NFU5 are most probably specific to the mitochondria (Leon *et al.*, 2003). The plant-specific NFU1/2/3 are composed of two NFU domains, the most C-terminal one having lost the CXXC motif involved in Fe-S cluster ligation. They differ from mitochondrial NFU4/5 that are also bimodular proteins formed by an N-terminal domain of unknown function associated to a C-terminal NFU domain (Couturier *et al.*, 2013). While NFU is an essential gene in *Synechococcus* and in human (Balasubramanian *et al.*, 2006; Navarro-Sastre *et al.*, 2011), this is not the case in *Azotobacter vinelandii*, *E. coli* and *Saccharomyces cerevisiae* although an *A. vinelandii nfuA* mutant is lethal under elevated oxygen concentrations (Angelini *et al.*, 2008; Bandyopadhyay *et al.*, 2008b; Melber *et al.*, 2016; Uzarska *et al.*, 2016). Concerning plants, the *A. thaliana nfu2* and *nfu3* mutants exhibit dwarf phenotypes, primarily explained by an impaired PSI accumulation and thus a deficient photosynthesis (Nath *et al.*, 2016; Touraine *et al.*, 2004; Yabe *et al.*, 2004). This raises notably some questions about the role of NFU1, about the respective contribution of these NFUs for PSI assembly relatively to HCF101 and about the existence of additional specific functions that would explain the presence of two to four plastidial NFU members in angiosperms. These questions have been addressed in this study focusing on the three plastidial *A. thaliana* NFUs by combining genetic, physiological, cellular and biochemical approaches.

## Material and methods

All PCR products were obtained using high-fidelity DNA polymerase and each construct was sequenced to ensure its integrity. All primers used are described in Supplementary Table 1.

## Plant materials

The *Arabidopsis thaliana nfu2-1* (SALK_039254C), *nfu2-2* (SALK_011376C), *nfu3-1* (GK-381H10), *nfu3-2* (GK-79C01) mutants were previously described (Nath *et al.*, 2016; Touraine *et al.*, 2004; Yabe
et al., 2004). The *nfu1-1* (GK-661F04) mutant was obtained from the Arabidopsis Biological Resource Center (ABRC). All mutants are in the Columbia (Col-0) accession, which was used as wild-type. For complementation, the full-length sequences of *NFU2* and *NFU3* were cloned into the pBS-35SLUC+ under the control of a 35S promoter (Ravet *et al.*, 2012), the whole cassettes excised by Sall/XbaI restriction and subcloned into the pBIBhygro binary vector. *Agrobacterium tumefaciens* MP90 was used to transform *A. thaliana* Col-0, *nfu2-1* and *nfu3-2* mutants using the floral dip method and transgenic plants selected by hygromycin resistance.

For metabolomic analyses, plants were cultivated in hydroponic conditions as described in (Fourcroy *et al.*, 2016). When grown on soil (Neuhaus Humin Substrat N2), plants were sown in pots as single individuals and irrigated with water. In all cases, they were grown in a controlled growth chamber under short-day (8h light/16h dark photoperiod) or long-day (16h light/8h dark photoperiod) conditions at a light intensity of 160 µmol m⁻² s⁻¹ and a relative air humidity of 70%. For chloroplast isolation and establishment of 77K chlorophyll fluorescence spectra, plants were grown in a phytotron under controlled conditions of light (PFD, 160 µmol m⁻² s⁻¹; day/night, 8h/16h), temperature (23°C/18°C, day/night) and relative air humidity (60%).

For root growth analyses, sterilized seeds are germinated on Petri dishes with a solidified half-strength Murashige and Skoog medium supplemented or not with 1% sucrose or with branched-chain amino acids (BCAA, Val, Ile, Leu) at a final concentration of 10 or 100 µM. After 48h at 4°C in darkness, plants were vertically grown for 7 days or longer in a growth chamber under long-day conditions.

## Separation of pigmented photosynthetic complexes

Stacked thylakoid membranes were prepared as described in (Caffarri *et al.*, 2009), but stopping the preparation before the Triton solubilizing step used for grana membrane preparation. Membranes were solubilised and fractionated as in (Caffarri *et al.*, 2009). In brief, 150 µg of thylakoids (on a Chl basis) were washed in 10 mM Hepes-KOH pH 7.5, resuspended in the same buffer at 1 mg/ml and then solubilized at a final concentration of 0.5 mg/ml by adding 1 volume of 0.6% (w/v) α-dodecylmaltoside (α-DDM) in 10 mM Hepes-KOH pH 7.5. Samples were vortexed a few seconds, centrifuged at 12,000 g for 10 min to remove insoluble material and then the supernatant was loaded on the top of a sucrose gradients formed directly in the tubes by freezing/thawing a solution of 0.6 M sucrose, 10 mM Hepes-KOH pH 7.5, 0.01% α-DDM. Ultracentrifugation was performed at 4°C for 18h at 41,000 rpm in a SW41 rotor.

## Chlorophyll fluorescence
Steady-state 77K fluorescence emission spectra have been measured on thylakoids resuspended in 85% (w/v) glycerol, 10 mM Hepes-KOH pH 7.5, by placing the sample in a glass Pasteur pipette and cooling with liquid nitrogen. Spectra have been measured using a Cary Eclipse fluorimeter (Varian) by exciting at 440 nm (slit 5 nm) and measuring fluorescence emission between 600 and 800 nm (slit 2.5 nm).

Chlorophyll fluorescence emission from attached leaves was measured with a FluorCam 7 (Photon System Instrument). The maximal quantum yield of PSII photochemistry was measured on dark-adapted plants as Fv/Fm=(Fm-Fo)/Fm where Fm is the maximum fluorescence level obtained with a pulse of intense white light and Fo is the initial fluorescence level in the dark. The actual quantum yield of PSII photochemistry ($\Phi_{PSII}$) was measured in the light as 1-(Fs/Fm') where Fs is the actual (steady-state) fluorescence level and Fm' is the maximal level in the light. $q_p$ was calculated with the formula: (Fm'-Fs)/(Fm'-Fo') where Fo' is the initial level in the light. $1-q_p$ is an indicator of the reduction level of the primary (quinone) PSII electron acceptor $Q_A$.

## Chloroplast ultrastructure

Small leaves were fixed in Sorensen 0.1 M buffer, pH 7.4 containing 4% glutaraldehyde for 4h at room temperature with vacuum infiltration, then overnight at 4°C. Then samples were rinsed in Sorensen buffer and post-fixed in 0.5% osmic acid in water for 2h at room temperature in the dark. After two washes in water, the samples were placed in a microwave inclusion automaton (Leica, AMW). They were dehydrated in a series of baths of ethanol (30-100%) and absolute acetone. Then, by means of acetone-resin progressive baths, the samples were coated in an epoxy resin: EMBed 812, polymerized at 60°C. Ultra-thin cuts (around 70 nm; Leica-Reichert Ultracut E) were then collected on copper grids at different levels of the samples. Then, they were contrasted in a solution of Oolong tea extract 0.2% in water for 20 min followed by 3 washes in water and observed using a Hitachi 7100 transmission electron microscope equipped with a Hamamatsu ORCA-SR digital camera.

## Enzymatic activities

About 100 to 150 mg of leaves were ground in liquid nitrogen and suspended in 250 µL of extraction buffer (100 mM Tris-HCl pH 7.8, 1 mM PMSF). Samples were kept on ice for 15 min, vortexed regularly and centrifuged at 13,000 g for 10 min. The supernatant was recovered and used for SiR and NiR activities as described in (Takahashi *et al.*, 2001; von Arb and Brunold, 1983).

## Protein extraction and Immunoblot analyses

Soluble proteins were obtained after centrifugation of the liquid nitrogen ground powder of *Arabidopsis* organs and resuspension in 100 mM Tris-HCl pH 8.0, 1% lithium dodecyl sulfate, 25 mM EDTA pH 8.0, 5% β-mercaptoethanol (P/V ratio=2.5), separation on 13% SDS-PAGE and transfer on
PVDF membranes. Rabbit polyclonal antibodies were raised against N-terminal His-tagged NFU1, NFU3, APR1 and untagged NFU2 and FDX2 by the GeneCust company (Luxembourg). The FTR (1/5000 dilution), PetC (1/10000) and PsaD (1/10000) were raised against the native spinach enzyme and *Chamydomonas* proteins respectively (gift from P. Schürmann, Neuchâtel, Switzerland and FA. Wollmann IBPC, Paris, France). The PsbA (1/20000) and Fd-GOGAT (1/10000) antibodies were from Agrisera and the NDH-H (1/10000) antibodies were a gift from D. Rumeau (CEA Cadarache, France). The molecular mass marker is the PageRuler Prestained Protein Ladder (Thermo Scientific).

Immunoblots on Lhca proteins were performed after separation of proteins on SDS-PAGE 15.5% with acrylamide/bis-acrylamide ratio of 75:1 in presence of 6 M urea and transfer of the proteins on nitrocellulose, using antibodies from Agrisera. Gels were replicated 3 times and loaded with the same amount of chlorophylls for each sample (0.4, 0.8 and 1.6 µg in the three repetitions).

## Heterologous expression in *E. coli* and purification of recombinant proteins

The sequences coding for the presumed mature forms of *A. thaliana* NFU1, NFU2, NFU3 were respectively cloned into pET15b, pET3d and a modified pET3c integrating N-terminal 6-His tag. Protein expression was achieved in the *E. coli* BL21(DE3) strain containing the pSBET plasmid (kanamycin resistance). Cultures were performed in LB medium at 37°C supplemented with the adequate antibiotics at 50 µg/mL. Protein expression was induced at exponential phase by adding 100 µM isopropyl β-D-thiogalactopyranoside for 4 h at 37°C. After centrifugation, the cell pellets were resuspended in 50 mM phosphate pH 8.0, 300 mM NaCl, 10 mM imidazole for his-tagged proteins, or 30 mM Tris-HCl pH 8.0, 1 mM EDTA, 200 mM NaCl for untagged proteins. After cell lysis, the soluble and insoluble fractions were separated by centrifugation for 30 min at 27,000 g. The His-NFU1 and His-NFU3 recombinant proteins were purified on Ni²⁺ affinity columns (Sigma-Aldrich) under native or denaturing conditions (8M urea) respectively following manufacturer's instructions. The purification of untagged NFU2 was carried out as in (Couturier et al., 2014). All recombinant proteins are stored in a 30 mM Tris-HCl pH 8.0 buffer at -20°C.

## Binary yeast two-hybrid assays

All experiments were performed in the Gal4-based yeast two-hybrid reporter strain CY306 (MATα, *ura3-52*, *his3-200*, *ade2-101*, *lys2-801*, *leu2-3*, *112*, *trp1-901*, *gal4-542*, *gal80-538*, *lys2::UASGAL1-TATAGAL1-HIS3*, *URA3::UASGAL4,17MERS(x3)-TATACYC1-LacZ*, *trx1::KanMX4*, *trx2::KanMX4*) (Vignols et al., 2005). The sequences coding for the mature form of each protein (same cleavage site as the one used for recombinant proteins) were cloned in both pGADT7 and pGBKT7 vectors (Clontech) in order to test binary interactions using both AD (Gal4 Activator domain) and BD (Gal4 DNA-binding domain) fusion combinations. CY306 cells co-transformed by pGAD and pGBK
constructs were grown on minimal YNB medium (0.7% yeast extract w/o amino acids, 2% glucose, 2% agar) with required amino acids (His, Ade, Lys, Ura, Met). Interactions were visualized as cells growing on YNB medium in the absence of histidine and in the presence of 3-aminotriazole (3AT). The 3AT concentrations used to evaluate the strength of each interaction assayed were determined after recording *HIS3* gene transactivation by individual proteins produced either as AD- or BD-fusions co-transformed with the empty partner vector. At least four independent transformations per binary assay were performed, of which 2 to 4 colonies were assayed. Images were taken 4 days post-d dotting (7 µL per dot at an optical density of 5.10⁻² at 600 nm).

## Bimolecular fluorescence complementation assay

Full-length open reading frames coding for NFUs, HCF101, and DHAD were cloned into both pUC-SPYNE and pUC-SPYCE vectors (Walter *et al.*, 2004) in the 5' ends of sequences coding for the N- and C-terminal regions of YFP. Arabidopsis protoplasts were prepared and co-transfected with pUC-SPYNE and pUC-SPYCE construct pairs for 5 min in a PEG-based medium as described in (Yoo *et al.*, 2007) without vacuum infiltration. Pairs of constructs involving one empty vector were also transfected to control that none of the protein assayed could restore YFP fluorescence in the absence of interacting partners. The YFP fluorescence in Arabidopsis protoplasts was recorded between 520 and 550 nm with a SP8 laser scanning confocal microscope (Leica Microsystems, Wetzler, Germany), after excitation with an argon laser at 514 nm. The chlorophyll auto-fluorescence was collected between 680 and 720 nm after excitation at 560 nm. The Leica LASX software was used to obtain images with maximum Z-stack intensity projection. Images were processed using the Adobe Photoshop software package. Results are representative of three independent bombardment experiments including the analysis of 10 to 20 cells per transformation event.

## Extraction, determination and quantification of metabolites by GC-MS

Metabolites from approximately 100 mg of ground frozen roots were extracted in a two-phase system of 2.5 ml methyl-*tert*-butyl ether (MTBE) and 1 ml H₂O, and the aqueous phase was re-extracted with 1.3 ml MTBE as described (Matyash *et al.*, 2008). *Allo*-inositol was used as an internal standard. 100 µl of the polar fraction was dried under a stream of nitrogen and derivatised with 15 µl methoxyamine hydrochloride in pyridine (30 mg/ml) and 30 µl N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) as previously described (Bellaire *et al.*, 2014) to transform metabolites into their methoxyimino (MEOX)- and trimethylsilyl (TMS)-derivatives. Each sample was analysed twice, once with a longer gradient and a split of 1:10, and once with a shorter gradient and a split of 1:50 to analyse substances with a high abundance. The samples were analysed on an Agilent 5977N mass
selective detector connected to an Agilent 7890B gas chromatograph equipped with a capillary HP5-MS column (30 m x 0.25 mm; 0.25 µm coating thickness; J&W Scientific, Agilent). Helium was used as carrier gas (1 ml/min). The inlet temperature was set to 250°C and the temperature gradient applied was 70°C for 2 min, 70 – 325°C at 5 K/min (15 K/min in the short gradient) and 325°C for 2 min. Electron energy of 70 eV, an ion source temperature of 230°C, and a transfer line temperature of 280°C were used. Spectra were recorded in the range of 70-600 da/e (Popko *et al.*, 2016). Metabolites were identified by the use of external standards or spectral libraries (Kopka *et al.*, 2005). Masses used for quantification of the individual metabolites are depicted in Supplementary Table 2.
## Results

### The growth of *nfu2* and *nfu3* mutant lines is differentially affected under contrasted photoperiods

A growth retardation of the aerial parts of *nfu2* and *nfu3* mutants has been previously reported and attributed to photosynthesis defects (Nath *et al.*, 2016; Touraine *et al.*, 2004; Yabe *et al.*, 2004). However, *nfu2-1*, *nfu2-2*, *nfu3-1* and *nfu3-2* mutant alleles were studied independently and in different conditions and no *nfu1* mutant was described. We have screened insertion mutant lines available for *NFU1* gene and selected the sole (*nfu1-1*) where *NFU1* is not detectable by immunoblot in a leaf protein extract (Fig. 1A) using antibodies raised against the corresponding recombinant protein. Additionally, we have evaluated the expression of all three NFUs in soluble protein extracts prepared from six organs of *Arabidopsis* wild-type plants. The anti-NFU1 and -NFU2 sera detected the corresponding proteins at the molecular mass expected for the predicted mature forms (*ca* 17-19 kDa). The NFU3 serum recognized both NFU2 and NFU3 but the latter is detected at around 14 kDa instead of the expected 17 kDa (Fig. 1A, Supplementary Fig. 1). This atypical migration was unexpected considering that NFU2 and NFU3 share 73% identity for their predicted mature part. Searching in proteomic databases, we determined that the cleavage site of the chloroplastic targeting sequence of NFU3 is before Val81 (Supplementary Fig. 2), which would correspond to a theoretical molecular mass of 16.7 kDa for the mature protein. Coupled to the detection of a peptide corresponding to the C-terminal end, we concluded that a particular N- or C-terminal end processing should not be the cause of the atypical migration observed. This incidentally suggested that NFU2 and NFU3 could have different properties despite their high similarity. Concerning the expression pattern, all NFUs are present in all organs tested, except roots, where NFU2 is clearly predominant because only trace amounts of NFU1 but not of NFU3 have been detected (Fig. 1A).

With these information about protein distribution patterns, we sought to examine the growth of *nfu* mutants in comparable conditions *i.e.*, on soil under short- or long-day conditions. For *nfu2* and *nfu3* allelic series, only one allele for each gene was investigated in detail because they exhibited the same growth phenotypes (Supplementary Fig. 3). When compared with Col-0, *nfu1-1* growth was similar in both photoperiod conditions (Fig. 1B, Supplementary Fig. 4). Under short-day conditions, *nfu2-1* and *nfu3-2* exhibited a strong alteration of growth,
but *nfu3-2* was much more affected (Fig. 1B). Under long-day conditions, *nfu2-1* and *nfu3-2* exhibited a much similar delayed growth suggesting that long days were either detrimental for *nfu2-1* or beneficial for *nfu3-2* (Fig. 1B).

To determine what could be the source of the differences observed between short- and long-day conditions for *nfu2-1* and *nfu3-2* mutant lines, photosynthetic parameters of Col-0, *nfu2-1* and *nfu3-2* lines were monitored on attached leaves (Fig. 1C&D, Supplementary Fig. 5). The impact of *nfu* mutations on the maximum quantum yield of PSII photochemistry (Fv/Fm) was very moderate (Fig. 1C), suggesting little effects on PSII. In contrast, photosynthetic electron transport (ΦPSII) in the light was strongly inhibited in leaves of both mutants (Fig. 1D). Moreover, the values of the 1-qP parameter were much higher in *nfu2-1* and *nfu3-2* relative to that of Col-0 (Supplementary Fig. 5). This indicates a marked accumulation of reduced quinone acceptors of PSII in these mutants (Maxwell and Johnson, 2000). Taken together, the chlorophyll fluorescence measurements indicated an inhibition of the photosynthetic electron transport system downstream of PSII. The fact that the ΦPSII parameter was lower and the 1-qP higher in long-day conditions in *nfu2-1* mutant compared to short-day conditions whereas an opposite trend is observed for Col-0 and *nfu3-2* indicated that long-day conditions are detrimental for the *nfu2-1* mutant (Fig. 1D and Supplementary Fig. 5).

## NFU2 and NFU3 are required for a functional PSI

The affected photosynthetic parameters in both *nfu2-1* and *nfu3-2* mutants prompted us to investigate the chloroplast (ultra)-structure and functioning. Photosystems and light-harvesting complexes of Col-0, *nfu2-1* and *nfu3-2* were separated on a sucrose gradient (Fig. 2A, Supplementary Fig. 6). A strong decrease of PSI abundance was confirmed in both *nfu* mutants. An apparent small increase of LHCII and monomeric PSII was observed but this is due to the fact that the same amount of membranes of each sample has been solubilised on a chlorophyll basis. Hence, the lower amount of PSI in both *nfu* mutants resulted in a slightly higher amount of PSII complex in the gradients. Then, the abundance of some proteins of the photosynthetic apparatus was determined, PsaD and PsbA (components of photosystems I and II respectively), the Rieske (PetC) protein of the cytochrome b6/f complex and the NDH-H protein, a subunit of the chloroplastic NAD(P)H dehydrogenase complex that contains two Fe-S subunits (NDH-I, NDH-K), with the specific aim of assessing cyclic electron flow (Rumeau *et al.*, 2007). When compared with Col-0, the abundance of PsbA and NDH-H remained unchanged in both mutants whereas an increase of the Rieske protein was observed (Fig. 2B). However, the most dramatic effect was the strong decrease of PsaD, a result consistent with the decrease of PSI abundance and very likely with its disorganisation. To examine further these aspects, chlorophyll fluorescence emission spectra at low temperature (77K) of thylakoid preparations were measured (Fig. 2C). The peak at around 730 nm, indicative of PSI antennae, was reduced in both mutants, especially
*nfu3-2*, compared to the PSII fluorescence peak at 685 nm indicating a global reduction of PSI content. Moreover, the displacement of the peak to lower wavelengths suggested that the two red Lhcas in the antenna system, Lhca3 and/or Lhca4, are reduced. In the *nfu3-2* mutant, the shoulder around 670 nm also indicated the presence of disconnected Lhc antennae. To validate these assumptions, the abundance of Lhca1, 2, 3 and 4 proteins in thylakoids was determined (Fig. 2D). While Lhca3 was the most affected, all Lhcas were decreased in both mutants and *nfu3-2* exhibited the strongest decrease. Importantly, no alteration of chloroplast ultrastructure was observed in both mutants (Supplementary Fig. 7). This whole set of data indicated that both NFU2 and NFU3 participate to the maturation of PSI, very likely by contributing to the assembly of one or several Fe-S clusters within PsaA, B and C proteins.

## NFU2 and NFU3 act in concert with HCF101

As the *nfu3-2* phenotype was stronger than that of *nfu2-1* in short-day photoperiod conditions and because the *nfu2-1* mutant was more affected by long-day conditions, the question of the existence of specific targets for both proteins was asked. Hence, the protein abundance and/or enzymatic activity of additional proteins harbouring different types of clusters were comparatively investigated in Col-0, *nfu2-1* and *nfu3-2* grown under identical conditions (Fig. 3A-C). Among [Fe₄S₄] proteins, we have examined the levels of APS reductases (APRs) because APR1, one of the three APR isoforms, was shown to interact with NFU2 by yeast two-hybrid (Y2H) experiments (Gao *et al.*, 2013) and of FTR catalytic subunit because its abundance is modified in the *hcf101* mutant (Lezhneva *et al.*, 2004). Antibodies raised against APR1 recognized three close bands in the 40 kDa area likely corresponding to the APR1/2/3 isoforms. The level of these [Fe₄S₄] proteins was not or slightly modified in *nfu2-1* and *nfu3-2* mutants (Fig. 3A). The effect on siroheme-dependent enzymes was also investigated by measuring their activities. The nitrite reductase (NiR) activity was not altered in both *nfu* mutants when compared with Col-0 (Fig. 3B) whereas a significant alteration of sulfite reductase (SiR) activity was observed in *nfu2-1* but not in *nfu3-2* (Fig. 3C). The glutamate synthase (GOGAT) is an enzyme containing a [Fe₃S₄] cluster. When compared with Col-0, a substantial increase of the protein level was observed in both *nfu2-1* and *nfu3-2* (Fig. 3A). As a representative of [Fe₂S₂] proteins, the level of FDX2 was also assessed. It did not vary much in *nfu2-1* and was increased in *nfu3-2*. The latter increase could be interpreted as a compensation mechanism to optimize the recovery of electrons in conditions with limited active PSI.

Besides the effect on SiR activity observed in *nfu2-1*, these results suggest that NFU2 and NFU3 are not involved in the maturation of the other tested Fe-S proteins unless apoproteins are stable enough, precluding to observe variations in abundance. It might also be linked to compensation mechanisms between both NFUs or with other transfer proteins as HCF101, which also acts in the late maturation
steps, at least for Fe-S subunits of PSI (Lezhneva et al., 2004; Stockel and Oelmuller, 2004). However, the level of HCF101 was not particularly modified in both *nfu* mutants with only a slight increase in *nfu3-2* (Fig. 3A). To better delineate the connections between NFUs, HCF101 and the PSI subunit, PsaC, we performed binary Y2H assays (Fig. 3D and Supplementary Fig. 8). We first found that both NFU2 and NFU3 were able to interact with HCF101 and that HCF101 could also interact with PsaC. No interaction was visible with the three NFU/PsaC pairs. *In Planta*, BiFC assays also indicated that strong NFU3/HCF101 interaction occurs as fast as 5h post-protoplast transfection (16h for NFU2/HCF101, Supplementary Fig. 9A), leading to fluorescent punctuate patterns that typically resemble that of plastidial NFU-GFP (Leon et al., 2003) and of other plastidial proteins locating near the thylakoids, where their over-accumulation seem to provoke membrane deformation (Machettira et al., 2011). An interaction between NFU1 and HCF101 was never observed using both methods (Supplementary Fig. 9B). Overall, considering that PsaC abundance is drastically decreased in *nfu3* mutants (Nath et al., 2016), and the genetic evidence that a double *nfu2-1* x *nfu3-2* mutant is lethal (Supplementary Fig. 10) as is the *hcf101* mutant (Lezhneva et al., 2004; Stockel and Oelmuller, 2004), these results support the view that NFU2 and NFU3, but not NFU1, would act upstream HCF101 for PsaC maturation.

### NFU2 is crucial for a proper root development by participating to the maturation of an enzyme involved in the branched-chain amino acid synthesis

Given the different expression patterns of NFUs in roots, we investigated the root growth of *nfu* mutants. Using a standard medium (MS/2-Agar) in the absence of sucrose, we observed that both *nfu2-1* and *nfu3-2* mutants exhibited a strong reduction of root growth when compared with Col-0, of about 50% and 70% respectively (Supplementary Fig. 11A, B). When the medium was supplemented with sucrose, the *nfu3-2* root growth was almost completely restored, reaching 80% of the growth observed for Col-0. This indicates that the defects observed in conditions of sucrose deprivation were primarily linked to an impaired photosynthesis, which is unable to provide sugar(s) for the proper growth of roots. The behaviour of *nfu2-1* was different, as sucrose addition did not restore the root growth defect. All of this suggested the deficiency of specific Fe-S protein target(s) in *nfu2-1* roots. Note that for both mutants, adding sucrose had beneficial effects on shoot development (Supplementary Fig. 11C).

Among plastidial proteins harbouring an Fe-S cluster and expressed in roots, enzymes involved in the biosynthesis of branched-chain amino acids (BCAAs, valine, leucine, isoleucine) retained our attention. Indeed, mutants impaired in the expression of a
dihydroxyacid dehydratase (DHAD) have a defective root growth (Zhang *et al.*, 2015). In plants, DHAD is an $[Fe_2S_2]$ protein catalysing the dehydration of 2,3-dihydroxy-3-isovalerate or 2,3-dihydroxy-3-methylvalerate to 3-methyl-2-oxobutanoate or 3-methyl-2-oxopentanoate for the synthesis of valine and isoleucine respectively (Flint and Emptage, 1988). Hence, as an Fe-S cluster maturation defect in DHAD may be responsible for the growth default of *nfu2-1* mutant, the physical interaction with NFUs was investigated. Using binary Y2H, DHAD was found to interact with NFU2 whereas a weaker interaction was also detected with NFU3 (Fig. 4A, Supplementary Fig. 8). A validation of the NFU2-DHAD interaction in chloroplasts was obtained by BiFC experiments (Fig. 4B, Supplementary Fig. 12). All of this supports the recent *in vitro* demonstration that NFU2 is fully competent for the maturation of an active DHAD (Gao *et al.*, 2018). Interestingly, the root growth defect of *nfu2-1* is complemented by expressing NFU3 (Supplementary Fig. 13). indicating that both proteins have probably similar biochemical capacities in sustaining the maturation of the $[Fe_2S_2]$-containing DHAD, but that the absence of NFU3 in roots precluded any compensation.

To obtain further physiological evidence and to determine whether BCAAs improved the primary root growth of the *nfu2-1* mutant, they were supplied, alone or in combination, to plantlets from sowing in the standard MS-agar medium (Fig. 4C, D). In Col-0, the addition of these amino acids had little effect on root growth and rather led to a slight decrease at 100 µM of the three amino acids. On the contrary, the root growth of the *nfu2-1* mutant was significantly improved by adding a mixture of the three amino acids (up to +158% of *nfu2-1* root growth in control conditions). The supply of BCAAs proved to be beneficial also for the shoot development of the *nfu2-1* mutant as visible by the substantial growth restoration and assessed by measuring the improvement of the quantum yield of PSII photochemistry (Supplementary Fig. 14).

As an additional evidence of a BCAA synthesis defect in the *nfu2-1* mutant, a metabolomic profile of roots was carried on three week-old or two month-old plants grown under hydropony in long-day and short-day conditions respectively (Fig. 5, Supplementary Fig. 15-17, Supplementary Table 2). This analysis revealed that the BCAA precursors, 2,3-dihydroxy-3-isovalerate and 2,3-dihydroxy-3-methylvalerate, were specifically accumulated in the *nfu2-1* mutant when compared to Col-0 supporting a defect in DHAD activity. On the other hand, steady-state levels of free BCAAs do not seem to be affected or even increased, as is the case for Ile and Val in the *nfu2-1* mutant grown under long-day conditions. However, a lower flux through the pool of free BCAAs due to a lower synthesis rate could also lead to a lower
protein synthesis rate resulting in reduction of proliferation. Furthermore, the accumulation of intermediates might interfere with overall metabolism additionally slowing growth.

# DISCUSSION

## The plastidial NFUs are not physiologically and biochemically equivalent

Although NFU1 appears expressed in all organs even though at low level in roots, a *nfu1* mutant does not exhibit any visible growth difference under standard growth conditions, raising the question of its role. When grown on soil under the same conditions, the growth and photosynthetic parameters indicated that *nfu3* mutants were more affected than *nfu2* mutants under short-day conditions. Strikingly, this difference was less visible under long-day conditions, because this condition was detrimental to *nfu2* mutants as reflected by the Φ<sub>PSII</sub> and 1-qP parameters. These observations point to some functional differences between NFU2 and NFU3, which are also visible from the SDS-PAGE migration behavior. In fact, a phylogenetic analysis indicates that NFU1 clusters independently from NFU2 and NFU3 according to the presence of a single representative in the NFU1 and NFU2/3 clade in algae and non-vascular plants (Supplementary Fig. 18). Hence, after the appearance of *NFU2* and *NFU3* by duplication from an ancestral gene, novel independent functions may have been acquired by each gene product.

## The plastidial NFU2 and NFU3 act upstream of HCF101 for PSI assembly

Besides NFS2, SUFE1 and SUFBCD proteins acting in the early-steps of the SUF machinery and which are clearly essential for the maturation of all plastidial proteins (Hu *et al.*, 2017; Van Hoewyk *et al.*, 2007; Xu and Moller, 2006), NFU2, NFU3 and HCF101 were described as more specific late-acting contributors required for the maturation of [Fe₄S₄]-containing proteins. However, the connection between NFUs and HCF101 was never investigated, although a functional link is evident from their gene clustering in several bacterial phyla, notably bacteroidetes and chlorobiaceae. According to its role in PSI subunit assembly, it is worth noting that, contrary to *nfu2* and *nfu3* mutants, the *hcf101-1* mutant is lethal at the seedling stage unless sucrose is provided (Lezhneva *et al.*, 2004; Stockel and Oelmuller, 2004). In this mutant, the chloroplast ultrastructure is quite disturbed (i.e. lack of assimilatory starch, disorganized grana stacks) whereas it is unaffected in *nfu2* and *nfu3* mutants (Supplementary Fig. 7). The fact that a double *nfu2-1* x *nfu3-2* mutant is embryo-lethal (Supplementary Fig. 10) suggested that both NFUs could act upstream of HCF101. This was demonstrated by showing (i) that NFU2 and NFU3 physically interacted with HCF101 but not PsaC whereas HCF101 was able to interact with PsaC and (ii) that FTR accumulation was not affected in
both *nfu2* and *nfu3* mutants whereas its level is decreased by about 50% in the *hcf101-1* mutant (Lezhneva *et al.*, 2004). Considering that the FTR apoform is unstable, the authors hypothesized that there is an HCF101-independent pathway to explain the 50% mature FTR remaining. Hence, having either NFU2 or NFU3 may be enough to provide the Fe-S cluster necessary for HCF101 to ensure the maturation of about 50% of FTR. Whether they are also implicated in the HCF101-independent pathway and could interact with a protein having a redundant function with HCF101 cannot be determined at this stage but the FTR decrease observed in the *hcf101-1* mutant suggests that NFU2 or NFU3 cannot donate directly their cluster to FTR.

Not many other Fe-S proteins were examined in the *hcf101* mutants available. The levels of another [Fe₄S₄] protein, the 7-HYDROXYMETHYL CHLOROPHYLL a REDUCTASE involved in the chlorophyll a cycle, of the [Fe₃S₄]-GOGAT, of the [Fe₂S₂]-Rieske type protein, PetC and PHEOPHORBIDE a OXYGENASE (PAO) involved in the b6/f complex and in chlorophyll breakdown respectively and of the [Fe₂S₂]-containing FDX1 were unchanged (Hu *et al.*, 2017). A globally similar trend was observed in the *nfu2-1* mutant except that there might be slightly increased amounts of PetC, FDX1 and GOGAT (Hu *et al.*, 2017). We have had similar observations for these proteins in the *nfu2-1* mutant and a further increase in the *nfu3-2* mutant. This contrasts with the described 30% decrease of GOGAT in *nfu3* mutants which was used to claim that NFU3 is required for the maturation of the [Fe₃S₄] cluster in GOGAT (Nath *et al.*, 2016) and with the reported FDX2 decrease in *nfu2* mutants (Touraine *et al.*, 2004; Yabe *et al.*, 2004). Whether these differences are inherent to differences in the growth conditions used is unclear at this stage. Among other [Fe₄S₄] enzymes that we have specifically tested in the shoots of *nfu* mutants, there was basically no substantial change in the amounts of APR isoforms and NDH complex. Among FDX-dependent siroheme-containing isoforms, the only noticeable effect observed in this study is a decrease of SiR but not NiR activity in *nfu2-1* although SiR and NiR protein levels were previously reported as decreased in *nfu2* mutants (Yabe *et al.*, 2004). This decrease seems robust since it was already reported using *nfu2* plants with decreased FDX levels (Touraine *et al.*, 2004), likely excluding that this change is associated to a variation in FDX levels. Overall, this indicates that both NFU2 and NFU3 are involved in the maturation of common but also specific [Fe₄S₄] proteins in shoots. Because of their partial redundancy, the list of targets likely remains fragmentary in the absence of a viable *nfu2* *x* *nfu3* mutant.

**The plastidial NFU2 is required for a proper root growth by contributing to the synthesis of branched chain amino acids**

Looking to the phenotypes associated with plastidial Fe-S enzymes expressed in roots e.g. thiamin and lipoate synthases, isoprenoid biosynthetic enzymes (IspG/H) or dehydratases involved in the BCAA
synthesis (DHAD and IPMI) (Balk and Pilon, 2011), we noticed that the short root phenotype of *nfu2-1* may be reminiscent of *dhad* mutants (Zhang *et al.*, 2015). Accordingly, the *nfu2-1* mutant accumulates huge amounts of BCAA precursors and supplying the three BCAAs in the growth medium of *nfu2-1* germinating plants promotes an efficient growth rescue. In line with an implication of NFU2 in the assembly of the [*Fe*₂*S*₂] cluster found in DHAD, we have observed that NFU2 is able to physically interact with DHAD. Coupled to the observation that NFU2, but not other Fe-S cluster transfer proteins (GRXS14, GRXS16 and SUFA1), can specifically transfer *in vitro* an [*Fe*₂*S*₂] cluster to an apo-DHAD, this pointed to NFU2 serving as the physiological cluster donor of DHAD (Gao *et al.*, 2018). However, the fact that the rescue of the *nfu2-1* mutant by BCAA was not complete may indicate that NFU2, which is the predominant plastidial NFU in roots, has other client proteins in this organ. These should be direct clients as HCF101 is absent in roots (Stockel and Oelmuller, 2004). All of this makes a true difference compared to the functioning of the SUF machinery in shoots. Interestingly, the fact that NFU3 complemented the *nfu2* root phenotype and has the capacity to interact with DHAD indicates that it should also be able to integrate and transfer both [*Fe*₂*S*₂] and [*Fe*₄*S*₄] clusters as NFU2 (Gao *et al.*, 2013).

## NFU position within the SUF machinery: comparison with the bacterial and mitochondrial ISC machineries

In organisms other than plants, there is usually a single NFU representative, which acts as a late Fe-S transfer protein for the maturation of an exclusive subset of [*Fe*₄*S*₄] proteins. In *E. coli*, NfuA rather operates under oxidative stress conditions for the maturation of complex I Fe-S subunits and of aconitase (Py *et al.*, 2012). It can receive its [*Fe*₄*S*₄] cluster from both SUF and ISC scaffold complexes, namely SufBCD and IscU/HscAB, before transferring it to IscA or SufA proteins. In Human, *nfu1* patients present defects in lipoate synthesis and thus in enzymes dependent on this cofactor and in one or several complexes (I, II or III) of the mitochondrial respiratory chain depending on the patients (Cameron *et al.*, 2011; Navarro-Sastre *et al.*, 2011). In yeast, the deletion of *NFU1* results in lower lipoylation and aconitase and SDH activities, but the growth phenotype is mild except when the cells are grown on acetate-containing minimal media (Navarro-Sastre *et al.*, 2011; Uzarska *et al.*, 2016). In both yeast and Human, the current model is that *Isa1/2* proteins act upstream *Nfu1* notably because the set of proteins affected in mutants for these genes encompasses *Nfu1* targets (Melber *et al.*, 2016; Uzarska *et al.*, 2016). The situation for the SUF machinery in plants appears different and possibly closer to the *E. coli* situation. Indeed, a *sufa1* mutant has no growth phenotype (Yabe and Nakai, 2006) suggesting that SUFA1 should either act downstream of NFUs or in a separate pathway for the maturation of specific, non-essential Fe-S proteins. Hence, NFU2 and NFU3 should receive their cluster directly from the scaffold SUFBCD complex on which [*Fe*₄*S*₄] or [*Fe*₂*S*₂]
clusters can be directly built up (Fig. 6) (Blanc *et al.*, 2014). Whatever the type of Fe-S cluster assembled on SUFBCD, we have demonstrated *in vitro* the possibility of a conversion between $[Fe_4S_4]$ and $[Fe_2S_2]$ clusters on NFU2 (Gao *et al.*, 2013). This could actually be a key property in the conversion process in case where SUFBCD only assembles $[Fe_4S_4]$ clusters. On the other hand, we have obtained strong evidence for a tight relationship between NFU2 and/or NFU3 and HCF101 in shoots whereas NFU2 would be the terminal donor in the case of DHAD in roots. This function may be conserved across organisms. Indeed, the yeast dihydroxyacid dehydratase counterpart, namely Ilv3, which also bears a $[Fe_2S_2]$ cluster was recovered in multiple independent mass spectrometry analyses from cells expressing Nfu1-Strep fusions, although the interaction could not be confirmed by affinity capture upon co-expression of Ilv3-FLAG (Melber *et al.*, 2016). Moreover, the activity of the *Staphylococcus aureus* IlvD ortholog, which incorporates a $[Fe_4S_4]$, is decreased in a *nfu* mutant (Mashruwala *et al.*, 2015). Concerning the MRP family members, there are two additional HCF101 paralogs in plants. It is interesting to note that the mitochondrial INDH is also a very-late acting protein *a priori* exclusively required for complex I assembly by acting on the translation machinery (Wydro *et al.*, 2013) whereas the cytosolic NBP35 has a completely different role, forming the scaffold system of the CIA machinery (Bastow *et al.*, 2017; Bych *et al.*, 2008).

In conclusion, all these data highlight the existence of very specific systems depending on the subcellular compartments, organs or organisms considered. Concerning the plastidial SUF machinery, NFU2 and NFU3 contribute to the maturation of $[Fe_4S_4]$ PSI subunits by acting upstream of HCF101. In roots, in absence of NFU3, NFU2 participates as a direct partner to the maturation of the $[Fe_2S_2]$ cluster of a dihydroxyacid dehydratase. This points to the unexpected involvement of NFU2 for the maturation of both cluster types unlike other prokaryotic and eukaryotic NFU proteins that are specifically involved in the maturation of $[Fe_4S_4]$ proteins. Considering the low number of potential Fe-S cluster transfer proteins (SUFA1, GRXS14, GRXS16, NFU1-3, HCF101) relative to final acceptors (more than 30 plastidial proteins, most of them bearing $[Fe_4S_4]$ clusters), additional NFU targets likely await identification.

**Supplementary data**

Supplementary data are available at JXB online.

Table S1. Primers used in this study.

Table S2. Metabolic changes in roots - relative abundances of the metabolites.

Fig. S1. Expression levels of NFU proteins determined by immunoblots.

Fig. S2. Amino acid sequence alignment of the three plastidial NFU proteins of *Arabidopsis thaliana* showing peptides identified by mass spectrometry.

Fig. S3. Mutants for NFU genes encoding plastidial proteins.
Fig. S4. Rosette fresh weights of *nfu* mutants.

Fig. S5. Reduction levels of the primary electron acceptor (Q<sub>A</sub>) of PSII.

Fig. S6. Photosystem abundance and integrity in *nfu2-1* and *nfu3-2* mutants.

Fig. S7. Chloroplast ultrastructure in *nfu2-1* and *nfu3-2* mutants.

Fig. S8. Auto-activation properties of proteins tested in the *HIS3*-based Gal4 yeast two-hybrid assay.

Fig. S9. BiFC analyses of HCF101-NFU interactions.

Fig. S10. Dissected siliques from *nfu2-1*, *nfu3-2* and from a double mutant homozygous for *nfu2-1* and heterozygous for *nfu3-2*.

Fig. S11. Effect of sucrose on seedlings growth.

Fig. S12. BiFC analyses of DHAD-NFU2 interaction.

Fig. S13. Complementation of the root phenotype of *nfu2-1* by NFU2 and NFU3.

Fig. S14. Effect of BCAAs on the shoot phenotype of *nfu2-1*.

Fig. S15. Mass spectra of dihydroxyvalerates.

Fig. S16. Mass spectra of dihydroxycaproates.

Fig. S17. Mass spectrum and potential fragmentation pattern of an analyte potentially representing derivatized 2-oxo-3-hydroxyisovalerate.

Fig. S18. Phylogenetic tree of *NFU* genes.

## Acknowledgments

The authors wish to thank Carine Alcon and the Montpellier Rio-Imaging platform, Montpellier-France, for expertise and assistance in confocal microscopy and Sabine Freitag for sample preparation. TI and IF acknowledge the support of the Service Unit for Metabolomics and Lipidomics of the Goettingen Center for Molecular Biosciences (GZMB) at the University of Goettingen. This work was supported by the Agence Nationale de la Recherche as part of the "Investissements d'Avenir" program (ANR-11-LABX-0002-01, Lab of Excellence ARBRE) and by grant no. ANR-2013-BSV6-0002-01.

## Conflict of interest

The authors declare that they have no conflicts of interest with the content of this article.

## Author contribution

BT, FV, TI, JC, CD, IF, MH, NR, FG designed the research; BT, FV, JPT, TI, TD, HCW, CM, NB, SC, MH, NR performed research; BT, FV, TI, JC, CD, IF, MH, NR, FG analysed the data; FV, TI, MH, NR, FG wrote the paper.
References

Abdel-Ghany SE, Ye H, Garifullina GF, Zhang L, Pilon-Smits EA, Pilon M. 2005. Iron-sulfur cluster biogenesis in chloroplasts. Involvement of the scaffold protein CpIscA. *Plant physiology* **138**, 161-172.

Angelini S, Gerez C, Ollagnier-de Choudens S, Sanakis Y, Fontecave M, Barras F, Py B. 2008. NfuA, a new factor required for maturing Fe/S proteins in Escherichia coli under oxidative stress and iron starvation conditions. *The Journal of biological chemistry* **283**, 14084-14091.

Balasubramanian R, Shen G, Bryant DA, Golbeck JH. 2006. Regulatory roles for IscA and SufA in iron homeostasis and redox stress responses in the cyanobacterium Synechococcus sp. strain PCC 7002. *Journal of bacteriology* **188**, 3182-3191.

Balk J, Pilon M. 2011. Ancient and essential: the assembly of iron-sulfur clusters in plants. *Trends in plant science* **16**, 218-226.

Balk J, Schaedler TA. 2014. Iron cofactor assembly in plants. *Annual review of plant biology* **65**, 125-153.

Bandyopadhyay S, Gama F, Molina-Navarro MM, Gualberto JM, Claxton R, Naik SG, Huynh BH, Herrero E, Jacquot JP, Johnson MK, Rouhier N. 2008a. Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe-2S] clusters. *The EMBO journal* **27**, 1122-1133.

Bandyopadhyay S, Naik SG, O'Carroll IP, Huynh BH, Dean DR, Johnson MK, Dos Santos PC. 2008b. A proposed role for the Azotobacter vinelandii NfuA protein as an intermediate iron-sulfur cluster carrier. *The Journal of biological chemistry* **283**, 14092-14099.

Bastow EL, Bych K, Crack JC, Le Brun NE, Balk J. 2017. NBP35 interacts with DRE2 in the maturation of cytosolic iron-sulphur proteins in Arabidopsis thaliana. *The Plant journal : for cell and molecular biology* **89**, 590-600.

Bellaire A, Ischebeck T, Staedler Y, Weinhaeuser I, Mair A, Parameswaran S, Ito T, Schonenberger J, Weckwerth W. 2014. Metabolism and development - integration of micro computed tomography data and metabolite profiling reveals metabolic reprogramming from floral initiation to silique development. *The New phytologist* **202**, 322-335.

Bernard DG, Cheng Y, Zhao Y, Balk J. 2009. An allelic mutant series of ATM3 reveals its key role in the biogenesis of cytosolic iron-sulfur proteins in Arabidopsis. *Plant physiology* **151**, 590-602.

Blanc B, Clemancey M, Latour JM, Fontecave M, Ollagnier de Choudens S. 2014. Molecular investigation of iron-sulfur cluster assembly scaffolds under stress. *Biochemistry* **53**, 7867-7869.
Bych K, Netz DJ, Vigani G, Bill E, Lill R, Pierik AJ, Balk J. 2008. The essential cytosolic iron-sulfur protein Nbp35 acts without Cfd1 partner in the green lineage. *The Journal of biological chemistry* **283**, 35797-35804.

Caffarri S, Croce R, Breton J, Bassi R. 2001. The major antenna complex of photosystem II has a xanthophyll binding site not involved in light harvesting. *The Journal of biological chemistry* **276**, 35924-35933.

Caffarri S, Kouril R, Kereiche S, Boekema EJ, Croce R. 2009. Functional architecture of higher plant photosystem II supercomplexes. *The EMBO journal* **28**, 3052-3063.

Cameron JM, Janer A, Levandovskiy V, Mackay N, Rouault TA, Tong WH, Ogilvie I, Shoubridge EA, Robinson BH. 2011. Mutations in iron-sulfur cluster scaffold genes NFU1 and BOLA3 cause a fatal deficiency of multiple respiratory chain and 2-oxoacid dehydrogenase enzymes. *American journal of human genetics* **89**, 486-495.

Chahal HK, Dai Y, Saini A, Ayala-Castro C, Outten FW. 2009. The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer. *Biochemistry* **48**, 10644-10653.

Couturier J, Touraine B, Briat JF, Gaymard F, Rouhier N. 2013. The iron-sulfur cluster assembly machineries in plants: current knowledge and open questions. *Frontiers in plant science* **4**, 259.

Couturier J, Wu HC, Dhalleine T, Pegeot H, Sudre D, Gualberto JM, Jacquot JP, Gaymard F, Vignols F, Rouhier N. 2014. Monothiol glutaredoxin-BolA interactions: redox control of Arabidopsis thaliana BolA2 and SufE1. *Molecular plant* **7**, 187-205.

Flint DH, Emptage MH. 1988. Dihydroxy acid dehydratase from spinach contains a [2Fe-2S] cluster. *The Journal of biological chemistry* **263**, 3558-3564.

Fontecave M, Choudens SO, Py B, Barras F. 2005. Mechanisms of iron-sulfur cluster assembly: the SUF machinery. *Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry* **10**, 713-721.

Fourcroy P, Tissot N, Gaymard F, Briat JF, Dubos C. 2016. Facilitated Fe Nutrition by Phenolic Compounds Excreted by the Arabidopsis ABCG37/PDR9 Transporter Requires the IRT1/FRO2 High-Affinity Root Fe(2+) Transport System. *Molecular plant* **9**, 485-488.

Gao H, Azam T, Randeniya S, Couturier J, Rouhier N, Johnson MK. 2018. Function and maturation of the Fe-S center in dihydroxyacid dehydratase from Arabidopsis. *The Journal of biological chemistry* **293**, 4422-4433.

Gao H, Subramanian S, Couturier J, Naik SG, Kim SK, Leustek T, Knaff DB, Wu HC, Vignols F, Huynh BH, Rouhier N, Johnson MK. 2013. Arabidopsis thaliana Nfu2 accommodates [2Fe-2S] or [4Fe-4S] clusters and is competent for in vitro maturation of chloroplast [2Fe-2S] and [4Fe-4S] cluster-containing proteins. *Biochemistry* **52**, 6633-6645.

Gupta V, Sendra M, Naik SG, Chahal HK, Huynh BH, Outten FW, Fontecave M, Ollagnier de Choudens S. 2009. Native Escherichia coli SufA, coexpressed with SufBCDSE, purifies as a [2Fe-2S]
protein and acts as an Fe-S transporter to Fe-S target enzymes. *Journal of the American Chemical Society* **131**, 6149-6153.

Hu X, Page MT, Sumida A, Tanaka A, Terry MJ, Tanaka R. 2017. The iron-sulfur cluster biosynthesis protein SUFB is required for chlorophyll synthesis, but not phytochrome signaling. *The Plant journal: for cell and molecular biology* **89**, 1184-1194.

Kopka J, Schauer N, Krueger S, Birkemeyer C, Usadel B, Bergmuller E, Dormann P, Weckwerth W, Gibon Y, Stitt M, Willmitzer L, Fernie AR, Steinhauser D. 2005. GMD@CSB.DB: the Golm Metabolome Database. *Bioinformatics* **21**, 1635-1638.

Kushnir S, Babiychuk E, Storozhenko S, Davey MW, Papenbrock J, De Rycke R, Engler G, Stephan UW, Lange H, Kispal G, Lill R, Van Montagu M. 2001. A mutation of the mitochondrial ABC transporter Sta1 leads to dwarfism and chlorosis in the Arabidopsis mutant starik. *The Plant cell* **13**, 89-100.

Leon S, Touraine B, Ribot C, Briat JF, Lobreaux S. 2003. Iron-sulphur cluster assembly in plants: distinct NFU proteins in mitochondria and plastids from Arabidopsis thaliana. *The Biochemical journal* **371**, 823-830.

Lezhneva L, Amann K, Meurer J. 2004. The universally conserved HCF101 protein is involved in assembly of [4Fe-4S]-cluster-containing complexes in Arabidopsis thaliana chloroplasts. *The Plant journal: for cell and molecular biology* **37**, 174-185.

Lill R. 2009. Function and biogenesis of iron-sulphur proteins. *Nature* **460**, 831-838.

Lill R, Muhlenhoff U. 2008. Maturation of iron-sulfur proteins in eukaryotes: mechanisms, connected processes, and diseases. *Annual review of biochemistry* **77**, 669-700.

Machettira AB, Gross LE, Tillmann B, Weis BL, Englich G, Sommer MS, Koniger M, Schleiff E. 2011. Protein-induced modulation of chloroplast membrane morphology. *Frontiers in plant science* **2**, 118.

Mashruwala AA, Pang YY, Rosario-Cruz Z, Chahal HK, Benson MA, Mike LA, Skaar EP, Torres VJ, Nauseef WM, Boyd JM. 2015. Nfu facilitates the maturation of iron-sulfur proteins and participates in virulence in Staphylococcus aureus. *Molecular microbiology* **95**, 383-409.

Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D. 2008. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. *Journal of Lipid Research* **49**, 1137-1146.

Maxwell K, Johnson GN. 2000. Chlorophyll fluorescence--a practical guide. *Journal of experimental botany* **51**, 659-668.

Melber A, Na U, Vashisht A, Weiler BD, Lill R, Wohlschlegel JA, Winge DR. 2016. Role of Nfu1 and Bol3 in iron-sulfur cluster transfer to mitochondrial clients. *eLife* **5**.

Nath K, Wessendorf RL, Lu Y. 2016. A Nitrogen-Fixing Subunit Essential for Accumulating 4Fe-4S-Containing Photosystem I Core Proteins. *Plant physiology* **172**, 2459-2470.
Navarro-Sastre A, Tort F, Stehling O, Uzarska MA, Arranz JA, Del Toro M, Labayru MT, Landa J, Font A, Garcia-Villoria J, Merinero B, Ugarte M, Gutierrez-Solana LG, Campistol J, Garcia-Cazorla A, Vaquerizo J, Riudor E, Briones P, Elpeleg O, Ribes A, Lill R. 2011. A fatal mitochondrial disease is associated with defective NFU1 function in the maturation of a subset of mitochondrial Fe-S proteins. *American journal of human genetics* **89**, 656-667.

Popko J, Herrfurth C, Feussner K, Ischebeck T, Iven T, Haslam R, Hamilton M, Sayanova O, Napier J, Khozin-Goldberg I, Feussner I. 2016. Metabolome Analysis Reveals Betaine Lipids as Major Source for Triglyceride Formation, and the Accumulation of Sedoheptulose during Nitrogen-Starvation of Phaeodactylum tricornutum. *PloS one* **11**, e0164673.

Py B, Gerez C, Angelini S, Planel R, Vinella D, Loiseau L, Talla E, Brochier-Armanet C, Garcia Serres R, Latour JM, Ollagnier-de Choudens S, Fontecave M, Barras F. 2012. Molecular organization, biochemical function, cellular role and evolution of NfuA, an atypical Fe-S carrier. *Molecular microbiology* **86**, 155-171.

Ravet K, Reyt G, Arnaud N, Krouk G, Djouani el B, Boucherez J, Briat JF, Gaymard F. 2012. Iron and ROS control of the DownSTream mRNA decay pathway is essential for plant fitness. *The EMBO journal* **31**, 175-186.

Roche B, Aussel L, Ezraty B, Mandin P, Py B, Barras F. 2013. Iron/sulfur proteins biogenesis in prokaryotes: formation, regulation and diversity. *Biochimica et biophysica acta* **1827**, 455-469.

Rumeau D, Peltier G, Cournac L. 2007. Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response. *Plant, cell & environment* **30**, 1041-1051.

Schaedler TA, Thornton JD, Kruse I, Schwarzlander M, Meyer AJ, van Veen HW, Balk J. 2014. A conserved mitochondrial ATP-binding cassette transporter exports glutathione polysulfide for cytosolic metal cofactor assembly. *The Journal of biological chemistry* **289**, 23264-23274.

Schwenkert S, Netz DJ, Frazzon J, Pierik AJ, Bill E, Gross J, Lill R, Meurer J. 2009. Chloroplast HCF101 is a scaffold protein for [4Fe-4S] cluster assembly. *The Biochemical journal* **425**, 207-214.

Stockel J, Oelmuller R. 2004. A novel protein for photosystem I biogenesis. *The Journal of biological chemistry* **279**, 10243-10251.

Takahashi M, Sasaki Y, Ida S, Morikawa H. 2001. Nitrite reductase gene enrichment improves assimilation of NO(2) in Arabidopsis. *Plant physiology* **126**, 731-741.

Touraine B, Boutin JP, Marion-Poll A, Briat JF, Peltier G, Lobreaux S. 2004. Nfu2: a scaffold protein required for [4Fe-4S] and ferredoxin iron-sulphur cluster assembly in Arabidopsis chloroplasts. *The Plant journal : for cell and molecular biology* **40**, 101-111.

Uzarska MA, Nasta V, Weiler BD, Spantgar F, Ciofi-Baffoni S, Saviello MR, Gonnelli L, Muhlenhoff U, Banci L, Lill R. 2016. Mitochondrial B011 and B013 function as assembly factors for specific iron-sulfur proteins. *eLife* **5**.
Van Hoewyk D, Abdel-Ghany SE, Cohu CM, Herbert SK, Kugrens P, Pilon M, Pilon-Smits EA. 2007. Chloroplast iron-sulfur cluster protein maturation requires the essential cysteine desulfurase CpNifS. *Proceedings of the National Academy of Sciences of the United States of America* **104**, 5686-5691.

Vignols F, Brehelin C, Surdin-Kerjan Y, Thomas D, Meyer Y. 2005. A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteins in vivo. *Proceedings of the National Academy of Sciences of the United States of America* **102**, 16729-16734.

von Arb C, Brunold C. 1983. Measurement of ferredoxin-dependent sulfite reductase activity in crude extracts from leaves using O-acetyl-L-serine sulfhydrylase in a coupled assay system to measure the sulfide formed. *Analytical biochemistry* **131**, 198-204.

Waller JC, Ellens KW, Alvarez S, Loizeau K, Ravanel S, Hanson AD. 2012. Mitochondrial and plastidial COG0354 proteins have folate-dependent functions in iron-sulphur cluster metabolism. *Journal of experimental botany* **63**, 403-411.

Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C, Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K, Kudla J. 2004. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. *The Plant journal : for cell and molecular biology* **40**, 428-438.

Wollers S, Layer G, Garcia-Serres R, Signor L, Clemancey M, Latour JM, Fontecave M, Ollagnier de Choudens S. 2010. Iron-sulfur (Fe-S) cluster assembly: the SufBCD complex is a new type of Fe-S scaffold with a flavin redox cofactor. *The Journal of biological chemistry* **285**, 23331-23341.

Wydro MM, Sharma P, Foster JM, Bych K, Meyer EH, Balk J. 2013. The evolutionarily conserved iron-sulfur protein INDH is required for complex I assembly and mitochondrial translation in Arabidopsis [corrected]. *The Plant cell* **25**, 4014-4027.

Xu XM, Moller SG. 2006. AtSufE is an essential activator of plastidic and mitochondrial desulfurases in Arabidopsis. *The EMBO journal* **25**, 900-909.

Yabe T, Morimoto K, Kikuchi S, Nishio K, Terashima I, Nakai M. 2004. The Arabidopsis chloroplastic NifU-like protein CnfU, which can act as an iron-sulfur cluster scaffold protein, is required for biogenesis of ferredoxin and photosystem I. *The Plant cell* **16**, 993-1007.

Yabe T, Nakai M. 2006. Arabidopsis AtIscA-I is affected by deficiency of Fe-S cluster biosynthetic scaffold AtCnfU-V. *Biochemical and biophysical research communications* **340**, 1047-1052.

Yoo SD, Cho YH, Sheen J. 2007. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. *Nature protocols* **2**, 1565-1572.

Zhang C, Pang Q, Jiang L, Wang S, Yan X, Chen S, He Y. 2015. Dihydroxyacid dehydratase is important for gametophyte development and disruption causes increased susceptibility to salinity stress in Arabidopsis. *Journal of experimental botany* **66**, 879-888.
**Present address:**

¶ Department of biological Sciences and Technology, National University of Tainan, 70005 Tainan, Taiwan.
Legends to the figures

<caption>**Figure 1.** Plastidial NFU protein expression profiles in *Arabidopsis* organs, and shoot phenotypes of *nfu* mutants.</caption>

(A) Expression profile of NFU proteins determined by immunoblot. These representative immunoblots were performed using 20 µg of soluble proteins extracted from different organs i.e., Root (R), True Leaf (TL), Floral Stalk (FS), Cauline Leaf (CL), Flower (F) and green Siliques (S) of wild type (Col-0) plants or from true leaves of *nfu1-1*, *nfu2-1* or *nfu3-2* mutants. Plants were grown for 30 days on a hydroponic medium under long-day photoperiod (16 hours light). The closest band of molecular mass marker (in kDa) is indicated on the left. Loading control (Ponceau S membrane staining) and uncropped immunoblot membranes are shown as Supplementary Fig. 1A-C.

(B) Effect of photoperiod on the growth of *nfu1-1*, *nfu2-1* and *nfu3-2* mutants. Col-0 and *nfu* mutants were grown on soil in growth chambers in short (8 hours light) or long days (16 hours light), with a light intensity of 160 µmoles.m⁻².s⁻¹. These pictures are representative of 26 day-old plants.

(C) Maximal efficiency of PSII photochemistry (Fv/Fm) in leaves of Col-0, *nfu2-1* and *nfu3-2* plants. White bars, long-day photoperiod; grey bars, short-day photoperiod. The values are the means ± SE of at least 9 measurements. Different letters indicate significant differences (Student's *t*-test: *P* <0.001).

(D) Quantum yield of PSII photochemistry (ΦPSII) in leaves of Col-0, *nfu2-1* and *nfu3-2* plants exposed to different photon flux density (PFDs) of white light. Representative curves for Col-0 (circles); *nfu2-1* (squares); *nfu3-2* (triangles) plants grown under short-day (gray, dotted lines) or long-day conditions (white, solid lines).

<caption>**Figure 2.** Photosystem I architecture and abundance in *nfu2-1* and *nfu3-2* mutants.</caption>

(A) Fractionation of solubilized thylakoids of Col-0 and *nfu* mutants by ultracentrifugation on sucrose gradients. The bands corresponding to PSI, LHCII trimeric and monomeric PSII have been indicated. Identification of bands is according to previous works (Caffarri *et al.*, 2001; Caffarri *et al.*, 2009) and confirmed by SDS-PAGE, native gel and absorption spectra (Supplementary Fig. 6).

(B) Immunoblot analysis of PsbA (subunit A of photosytem II), PsaD (subunit D of photosystem I), PetC (Photosynthetic Electron Transfer C) and NDH-H (NAD(P)H-quinone oxidoreductase subunit H) abundance in adult leaves of *Arabidopsis* plants grown for 3 weeks after sowing under long-day conditions. These representative immunoblots were performed using 20 µg of soluble proteins. The closest band of molecular mass marker (in kDa) is indicated on the left.

(C) Chlorophyll fluorescence emission spectra at 77K of thylakoid preparations from Col-0 (continuous line), *nfu2-1* (dotted line) and *nfu3-2* (point line). Spectra have been normalised relatively to PSII maximum emission (685 nm).
(D) Immunoblot analyses for Lhca1 (black), 2 (white), 3 (pale grey) and 4 (grey) proteins in thylakoids. Gels were loaded with the same amount of thylakoid (on a chlorophyll basis). A Sypro-stained gel and a membrane coloured with Ponceau S are shown as loading controls for the gels (region containing Lhc proteins, 20-30 kDa). A relative increase of LHCII is visible in thylakoids from mutant plants. In the lower panel, the average values ± SE (three replicate immunoblots) of the Lhca's content in mutant lines normalized to the content in Col-0 are shown. Decreases of Lhca proteins in mutant plants as compared with Col-0 are statistically significant (*P* < 0.02).

<caption>**Figure 3. Activity and protein abundance of putative NFU2 or NFU3 client proteins in shoots of *nfu2-1* and *nfu3-2* mutants.**</caption>

(A) Immunoblot analysis of APS reductase 1 (APR1), ferredoxin-thioredoxin reductase (FTR), ferredoxin 2 (FDX2), glutamine-oxoglutarate aminotransferase (GOGAT) and high-chlorophyll fluorescence 101 (HCF101) relative abundance in adult leaves of *Arabidopsis* plants grown for 3 weeks after sowing in long-day conditions. The images are representative of immunoblots performed using three different protein extracts (Loading: 20 µg of soluble proteins). The closest band of molecular mass marker (in kDa) is indicated on the left.

(B) Nitrite reductase activity. The values expressed in nmol nitrite. mg⁻¹protein.min⁻¹ are the means ± SE (n=5). Identical letters indicate no significant differences (Student's *t* test: *P*<0.05).

(C) Sulfite reductase activity. The values expressed in nmol cysteine. mg⁻¹protein.min⁻¹ are the means ± SE (n=5). Different letters indicate significant differences (Student's *t* test: *P*<0.05).

(D) Binary interactions between plastidial NFUs, HCF101 and PsaC detected by yeast two-hybrid assays. Cells co-transformed with pGAD- and pGBK-based constructs (7 µL) were plated at an OD₆₀₀ of 5.10⁻² on a control plate containing histidine (+HIS) and on test plates without histidine (-his) in the presence of 5 mM 3-aminotriazol (-his + 3AT) when required depending on *HIS3* gene transactivation capacities exhibited by individual proteins (Supplementary Fig. 8). Yeast cells were grown at 30°C for 4 to 5 days. Results shown here are representative of 4 independent transformation experiments and correspond to those obtained using constructs expressing NFU fused to the Gal4 activator domain (AD) and potential interactors fused to the Gal4 DNA-binding domain (BD), except for HCF101 that exhibits *HIS3* gene transactivation (Supplementary Fig. 8). In this case, the result is shown for the interaction assayed with the opposite fusion proteins.

<caption>**Figure 4. Defects in branched-chain amino acid synthesis affect the root growth of the *nfu2-1* mutant.**</caption>

(A) Binary yeast two-hybrid experiments showing the interaction between NFUs and DHAD. Yeast cells were plated at an OD₆₀₀ of 5.10⁻² on a control plate containing histidine (+HIS) and on a test plate without histidine (-HIS) in the presence or not of 5 mM 3AT and grown at 30°C for 4 days. The left
panels show the yeast growth obtained with the AD-DHAD/BD-NFU pairs whereas the right panels show the yeast growth obtained with AD-NFU/BD-DHAD combinations. The results shown here are representative of three independent transformation experiments.

(B) YFP fluorescence images of *Arabidopsis* leaf protoplasts expressing DHAD fused to the C-terminal region of YFP (DHAD-C) and NFU2 fused to the N-terminal region of YFP (NFU2-N). YFP fluorescence was visible after 36 h and recorded after 48 h post-transfection. The scale bars correspond to 10 µm. The specificity of interactions between DHAD and NFU proteins was evaluated by replacing one of the two constructs in every couples assayed by an empty vector. None of these negative control experiments gave rise to YFP fluorescence (Supplemental Figure 12).

(C) The primary root growth phenotype of *nfu2-1* is rescued by branched-chain amino acids. Col-0 and *nfu2-1* mutant were sown on MS/2-Agar plates with 1% sucrose in the absence (0 µM) or presence (100 µM) of isoleucine, leucine, valine alone or in combination (10 µM or 100 µM) and grown under long-day conditions. The length of the primary root was measured after 7 days. The values are the mean ± SE (n=35). Identical letters indicate no statistical difference (Student's *t*-test: *P* < 0.001).

(D) Representative pictures showing the rescued root phenotype of 11 day-old plants. Scale bar 0.5 cm.

**Figure 5. Map of root metabolites.**

The plants were grown hydroponically either for two months under short-day conditions or for three weeks under long-day conditions. The metabolites extracted from root samples were derivatized with methoxyamine hydrochloride and N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed by GC-MS. Metabolites converted by enzymes that are potential targets of NFU2 are highlighted in blue as is OHIV, a further upstream metabolite. Error bars indicate SD; n=3. A star indicates a significant difference (*p*>0.01).

CoA, Coenzyme A; DHIV, (R)-2,3-Dihydroxy-isovalerate; DHMV, (R)-2,3-Dihydroxy-3-methylvalerate; GA3P, glyceraldehyde 3-phosphate; GABA, γ-aminobutyric acid; OHIV, 2-Oxo-3-hydroxyisovalerate; PEP, phosphoenolpyruvate; put. DHC, putative dihydroxycaproate; put DHV, putative dihydroxyvalerate; U18.6/19.29/20.53, unknown substances with a retention time of 18.6/19.29/20.53 min; A178003-101/A159003-101, unknown analytes deposited under these numbers in the Golm metabolome database.

**Figure 6. Positioning of NFUs and HCF101 in the current model of the SUF machinery and known client proteins.**

The NFS2 protein, assisted by SUFE proteins, provides the sulfur atom (yellow circle) that is combined with an iron atom (red circle) for the de novo assembly of a Fe-S cluster on the SUFBCD scaffold proteins. From the decreased abundance or activity in mutants (blue arrows) and binary yeast two hybrid experiments (red arrows), we propose that NFU2 and NFU3 serve as relays, receiving Fe-S clusters
from SUFBCD before transferring it to HCF101 in aerial parts. Known targets of HCF101 are Fe-S proteins belonging to PSI, PsaA, B and C and FTR (Lezhneva *et al.*, 2004). NFU2 likely possesses direct client proteins in the absence of HCF101 as shown for DHAD in roots. NFU1 has different, yet unknown, functions and partners. Other candidate SUF components *i.e.*, BOLA1/4, GRXS14, GRXS16, SUFA1 and IBA57.2 have not been represented here because their position is not yet defined.

### Accession numbers

Sequence data from this study can be found in The Arabidopsis Information Resource (TAIR) under the following accession numbers: NFS2 (AT1G08490), SUFE1 (AT4G26500), SUFB (AT4G04770), SUFC (AT3G10670), SUFD (AT1G32500), SUFA1 (AT1G10500), NFU1 (AT4G01940), NFU2 (AT5G49940), NFU3 (AT4G25910), HCF101 (AT3G24430), IBA57.2 (AT1G60990), GRXS14 (AT3G54900), GRXS16 (AT2G38270), BOLA1 (AT1G55805), BOLA4 (AT5G17560), DHAD (AT3G23940), PSAC (ATCG01060). Germplasms used are as follows: *nfu1-1* (GK-661F04), *nfu2-1* (SALK_039254C), *nfu2-2* (SALK_011376C), *nfu3-1* (GK-381H10), *nfu3-2* (GK-79C01).
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

Long
days

Short
days

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>Figure 2</figcaption></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>Figure 3</figcaption></figure>
A

<figure><img src="image_15.png" /></figure>

B

<figure><img src="image_16.png" /></figure>

C

<figure><img src="image_17.png" /></figure>

D

<figure><img src="image_18.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>Figure 5</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>Figure 6</figcaption></figure>