Crops utilize nitrogen (N) mainly in two different forms: nitrate (NO3) and ammonium (NH4+). N is one of the major components of chlorophyll, energy-transfer compounds such as ATP, nucleic acids, and proteins. Therefore, N availability in crops controls the rate of photosynthesis, cell growth, metabolism, and protein synthesis1. Crops are dependent on an adequate N supply, typically obtained from industrial synthetic fertilizers, but they do not assimilate more than half of the N applied as fertilizer, the remainder spilling over or leaching from the soil as a major source of pollution2,3,4. It is therefore important to explore strategies that will reduce the dependence of agriculture on N fertilizers.

Biological nitrogen fixation (BNF) is the reduction of N2 gas to ammonia (NH3) by the enzyme nitrogenase5. BNF is widespread in prokaryotes (bacteria and archaea), but no eukaryote species is yet known to directly convert N2 into a biologically useful form6. The direct transfer of nitrogenase genes from prokaryotes to crops is one of the most ambitious strategies to achieve BNF in plants7,8. Nitrogenases are composed of two O2-sensitive interacting metalloproteins: dinitrogenase and dinitrogenase reductase5. In the molybdenum (Mo) nitrogenase, which is the most abundant and best characterized form, these components are known as the MoFe protein (encoded by nifD and nifK) and Fe protein (encoded by nifH), respectively. In addition, multiple accessory components are required for nitrogenase assembly and activity9. The MoFe protein (NifD2K2) includes two P-clusters [8Fe-7S] and two FeMo-cofactors [7Fe-9S-C-Mo-R homocitrate], and its role is to bind and reduce N2. The Fe protein is a NifH homodimer that presents a single [4Fe-4S] cluster linking the two subunits and one site for MgATP binding and hydrolysis in each subunit10. The Fe protein is the obligate electron donor to the MoFe protein for N2 reduction11. It is also required for the maturation and activation of the MoFe protein P-cluster and FeMo-co9. Because the Fe protein is more sensitive to O2 than the MoFe protein, and because it is also required for the biosynthesis of MoFe protein cofactors, its functional expression has been used as proof of principle for nitrogenase engineering in eukaryotes12.

Mitochondria and chloroplasts are energy-conversion organelles in eukaryotic cells with the capacity to provide low potential electrons and ATP needed for BNF. The low-O2 environment of mitochondria also protects nitrogenase from O2 damage12. Both organelles have therefore been tested for the expression of nitrogenase Fe protein12,13,14,15. Active Fe protein was produced by expressing NifH and NifM as the minimal complement, optionally together with NifS and NifU depending on [Fe-S] cluster biosynthesis and insertion by the host species. NifM is a putative peptidyl prolyl cis‐trans isomerase that facilitates Fe protein folding and improves its solubility16,17. NifS mobilizes S from cysteine for the synthesis of [Fe-S] clusters on NifU, which subsequently donates them to cluster-less apo-Fe protein18,19. When NifH and NifM were co-expressed in the mitochondria of the yeast Saccharomyces cerevisiae, NifU and NifS were not required because the mitochondrial [Fe-S] cluster biosynthetic machinery was able to load [4Fe-4S] clusters onto the apo-NifH protein efficiently12. However, when NifH was co-expressed transiently with NifM, NifS and NifU in the mitochondria of Nicotiana benthamiana, the nitrogenase activity was lower than that achieved in yeast, suggesting that success of [4Fe-4S] cluster insertion ultimately depends on the [Fe-S] cluster machinery of the host15.

Here, we engineered rice to express selected nifH and nifM genes, targeting the corresponding proteins to the mitochondrial matrix to minimize O2 damage and produce correctly folded and enzymatically active NifH. Soluble NifH polypeptides accumulated in rice mitochondria and the as-isolated Fe protein showed limited functionality in electron donation to the MoFe protein and in FeMo-co synthesis, two fundamental activities to engineer nitrogenase. In vitro transfer of [4Fe-4S] clusters from the NifU donor to NifH was necessary to achieve maximum Fe protein activity. This result indicates that even though NifH incorporated some endogenous rice mitochondrial [4Fe-4S] clusters leading to activity, much of it accumulated as apo-protein, thereby identifying [Fe-S] cluster biosynthesis, insertion and stability as areas that should be the focus of future research.


Transformation and recovery of transgenic rice callus and plants expressing NifH and NifM targeted to the mitochondria

The genes encoding Hydrogenobacter thermophilus NifH (NifHHt) and Azotobacter vinelandii NifM (NifMAv) were cloned in separate vectors and introduced into rice along with the hygromycin phosphotransferase (hpt) gene for selection. The nifHHt and nifMAv sequences were previously codon optimized for S. cerevisiae and expressed in both S. cerevisiae and N. benthamiana15. The same codon-optimized nifHHt and nifMAv sequences were used because no rare codons were present in the two genes in the context of rice codon usage20. Expression of the nifHHt gene was driven by the strong constitutive maize ubiquitin promoter and first intron (ZmUbi1 + 1sti). The N-terminal 29-amino-acid mitochondrial transit peptide from S. cerevisiae cytochrome c oxidase subunit IV (Cox4) was added to the coding sequence, based on previous work showing its ability to direct GFP to rice mitochondria21. The 28-amino-acid Twinstrep (TS) tag was also added to facilitate NifHHt detection and purification. This tag did not affect NifH activity in A. vinelandii, S. cerevisiae or N. benthamiana14,15. An auxiliary DNA vector was constructed to co-express nifMAv under the control of the constitutive OsActin promoter. In this case, an N-terminal transit peptide from Neurospora crassa subunit 9 (Su9), was used because it was shown to deliver GFP to rice mitochondria21 and NifM to N. benthamiana mitochondria15. The process for the recovery of transgenic rice callus expressing nif transgenes and the regeneration of transgenic plants is shown in Supplementary Fig. 1.

Co-expression of OsNifHHt and OsNifMAv

The expression of OsNifHHt and OsNifMAv was analyzed at the mRNA and protein levels in three independent rice callus lines and the corresponding regenerated plants (Fig. 1 and Supplementary Fig. 2). Both genes were expressed at higher levels in plants than in the callus (Fig. 1a, b). The nifHHt gene was placed under a stronger promoter than nifMAv because the former encodes the most abundant Nif protein and it is part of the nitrogenase complex. Although there is no study proving that NifM is essential for NifH in plant mitochondria, NifM has been shown to be required for NifH solubility in heterologous expression systems including E. coli, yeast mitochondria, and plant chloroplasts14,16,22. Although we were unable to obtain clear western blot signals for OsNifMAv in the plant extracts, the fact that OsNifHHt was soluble indicated that OsNifMAv was expressed at sufficient levels to facilitate NifH polypeptide maturation, as recently shown for NifHHt in S. cerevisiae22. In callus, both OsNifHHt and OsNifMAv gave rise to bands of the expected size (~33 kDa for both proteins) when analyzed by SDS-PAGE and western blot, indicating that the proteins were correctly processed and stable in rice mitochondria (Fig. 1c, d). Analysis of OsNifHHt protein expression in the T1 generation confirmed that OsNifHHt was stably inherited and expressed in progeny (Fig. 1e, f).

Fig. 1: Expression of rice-derived OsNifHHt and OsNifMAv.
figure 1

Relative mRNA expression levels of OsnifHHt and OsnifMAv in three different (independent biological replicates) rice plant lines (a) and the corresponding callus lines (b). Data (normalized to OsActin mRNA) are means ± SD (n = 3 technical replicates). Immunoblot analysis of soluble protein extracts from rice leaves (c) and callus (d) probed with antibodies against NifH, NifM, and the Strep-tag. Antibodies against RuBisCO were used as loading control for plant lines. Ponceau staining was used as loading control for callus extracts due to the low expression of RuBisCO. Ctrl lane shows non-transformed callus and plant lines. e Stable expression of OsNifHHt in the T1 segregating generation of rice plant line HtH200. Protein extract from callus expressing OsNifHHt (line HtH206) was used as positive control (Pos ctrl). Uncropped immunoblots are shown in Supplementary Figs. 610. f Phenotype of OsNifHHt expressing T1 progeny showing normal growth and development.

Purification of OsNifHHt

OsNifHHt was purified from rice callus and the corresponding regenerated plants by strep-tag affinity chromatography (STAC) (Fig. 2a, b; Supplementary Fig. 3). To minimize O2 exposure produced during photosynthesis and maximize the isolation of functional OsNifHHt, plants were Fe-fertilized and harvested before onset of light at the end of the dark period, following a procedure previously shown to be successful to obtain active NifHAv and NifHHt from tobacco chloroplasts and mitochondria15,23. Purified OsNifHHt was mostly soluble and could be isolated from plant line Ht200 with yields of 0.5 mg kg−1 fresh weight (1-month-old plants) or 0.25 mg kg−1 fresh weight (2-month-old plants). The yield of OsNifHHt from three independent callus lines was ~2.5 mg kg−1 (line Ht189), ~7 mg kg−1 (line Ht200) and ~6.5 mg kg−1 (line Ht202) (Supplementary Fig. 4). Purified OsNifHHt from callus and plants migrated as a single major band indicating correct processing (Fig. 2a, b). The side-by-side comparison of ScNifHHt and OsNifHHt (both targeted to the mitochondria) was also consistent with the correct processing of OsNifHHt in the rice mitochondrial matrix, resulting in proteins with molecular weights of ~33 kDa (Fig. 2c). N-terminal sequencing of purified OsNifHHt revealed a major product with the anticipated N-terminal residues QKP following the cleavage of the Cox4 peptide, together with three minor variants representing alternative processing events (Fig. 2d). Mass spectrometry identified the OsNifHHt protein with 63% sequence coverage and confirmed that the C-terminus of the protein was intact (Supplementary Fig. 5).

Fig. 2: STAC purification of OsNifHHt protein targeted to rice mitochondria.
figure 2

a Purification of OsNifHHt from Ht189 callus. Fractions were analyzed by SDS-PAGE followed by Coomassie staining or immunoblotting. TE total extract, CFE soluble cell-free extract, FT flow-through fraction, W wash fraction, E elution fraction. Econc concentrated elution fraction (final sample collected). s.e. short exposure, l.e. long exposure. Uncropped immunoblot analysis for the purification processes are shown in Supplementary Fig. 11. b Final OsNifHHt sample isolated from Ht200 and Ht202 callus lines, and Ht200 rice plants. Uncropped Coomassie gels and immunoblot analysis for the purification processes are shown in Supplementary Fig. 3. c Side-by-side comparison of NifHHt migration when isolated from Ht189 callus (OsNifHHt) or yeast (ScNifHHt). d N-terminal sequencing of OsNifHHt isolated from Ht189 callus. The deduced Cox4 mitochondrial targeting peptide processing sites are indicated with arrows (the dominant processing size indicated by a larger arrow). The underlined sequences indicate peptide linkers between the Cox4 signal and the TS-tag (SAASA), and between the TS-tag and OsNifHHt (SS).

Purified OsNifHHt shows Fe protein activity

The Fe protein activity of purified OsNifHHt was determined in vitro using an acetylene reduction assay after mixing with NifDK purified from A. vinelandii (NifDKAv). The OsNifHHt protein isolated from rice leaves showed low activity (~7 nmol ethylene min−1 mg−1 NifDKAv) (Fig. 3a), probably limited by the relatively low yield of NifH in the plant tissue and hence the low amount of NifH (~8.9 µg) and low ratio of NifH:NifDK (~10:1) in the assay. To confirm the functionality of the as-isolated rice Fe protein, we performed titration experiments using increasing amounts of OsNifHHt purified from three independent callus lines. As anticipated for a functional Fe protein, higher activity was observed with an increasing NifH:NifDK ratio (Fig. 3b). Importantly, the activity of the OsNifHHt protein isolated from rice leaves was about half that of the callus protein at a similar ratio, which may reflect the use of more diluted plant OsNifHHt protein in the assay (we typically observed slight inhibition caused by large amounts of buffer).

Fig. 3: Acetylene reduction assay (ARA) with purified OsNifHHt.
figure 3

a ARA using OsNifHHt isolated from HtH200 rice plants (10:1 ratio of OsNifHHt:NifDKAv) (data are means ± SD, n = 2 technical replicates). Dotted line indicates the negative control ARA in the absence of NifH (mean of n = 3 technical replicates). The positive control ARA (40:1 ratio of NifHAv:NifDKAv) generated 1285 ± 225 units (mean ± SD, n = 3 technical replicates). b ARA with increasing amounts of OsNifHHt isolated from three different rice callus lines (0, 5:1, 10:1 and 20:1 ratio of OsNifHHt:NifDKAv). The positive control ARA (20:1 ratio of NifHAv:NifDKAv) generated 1426 ± 43 units for Ht189, 1127 ± 65 units for Ht200 and 1074 ± 153 units for Ht202 (data are means ± SD, n = 3 technical replicates). c ARA using OsNifHHt isolated from three different rice callus lines at a 40:1 ratio of OsNifHHt:NifDKAv. The positive control ARA (40:1 ratio of NifHAv:NifDKAv) generated 1285 ± 225 units (data are means ± SD, n = 3 technical replicates). d ARA using OsNifHHt and ScNifHHt before and after reconstitution with [4Fe-4S] cluster-loaded EcNifUAv. The positive control ARA (using NifHAv) generated 1553 ± 82 units. All reactions were performed with a 20:1 ratio of NifH:NifDKAv (data are means ± SD, n = 2 technical replicates). All activities are reported as nmol ethylene formed per min and mg of NifDKAv.

OsNifHHt appeared completely soluble in all three callus lines (Fig. 2a, Supplementary Fig. 3a, b), despite the varying expression level of NifM (Fig. 1b, d). The activities of these three OsNifHHt proteins in the same experiment at the same NifH:NifDK ratio of 40:1 were almost identical (Fig. 3c). This indicated that OsNifMAv expression was sufficient in all three rice lines, which was consistent with recent results obtained in S. cerevisiae22.

We then supplemented the reaction mixture with pure A. vinelandii NifU expressed in Escherichia coli and loaded with [4Fe-4S] clusters (EcNifUAv). NifU is known to serve as [4Fe-4S] donor to apo-NifH in vivo24 and in vitro19. Indeed, the reconstitution of OsNifHHt by the in vitro transfer of [4Fe-4S] from NifU increased its activity ~9-fold (compare activities in Fig. 3c, d). Importantly, the activity of OsNifHHt and ScNifHHt proteins was identical after reconstitution (Fig. 3d), suggesting that OsNifHHt was correctly folded but not fully mature, probably due to insufficient insertion of the [4Fe-4S] cluster into OsNifHHt in the rice mitochondria, or perhaps due to instability of the clusters as previously suggested in N. benthamiana15. These results demonstrate that the activities of reconstituted rice and yeast Fe protein are comparable.

Purified OsNifHHt supports in vitro FeMo-co synthesis

The isolated OsNifHHt also supported in vitro FeMo-co synthesis, which is another fundamental role of NifH required for BNF. FeMo-co synthesis and the activation of apo-NifDKAv (containing P-clusters but devoid of FeMo-co) was measured in vitro by combining Methanothermobacter thermautotrophicus NifB expressed in yeast (ScNifBMt), EcNifUAv (provider of [4Fe-4S] clusters for NifB-co biosynthesis), S-adenosyl methionine (SAM), molybdate, homocitrate, apo-NifENAv (isolated from a ΔnifB strain containing permanent [4Fe-4S] clusters but lacking the FeMo-co precursor), purified OsNifHHt and apo-NifDKAv.

The in vitro FeMo-co synthesis assay can be divided in three steps9. In the first step, NifB-co synthesis is catalyzed by NifB, which carries three [4Fe-4S] clusters that are provided by NifU25,26. This SAM-radical enzyme fuses two of its [4Fe-4S] clusters, incorporates an additional sulfide, and generates a carbide atom that is inserted at the center of this Fe-S frame. The result is an [8Fe-9S-C] called NifB-co that is then transferred to the NifEN scaffold to serve as FeMo-co precursor. In this step, NifU [4Fe-4S] clusters used as substrates for NifB are also likely to activate some of the cluster-less OsNifHHt protein. In the second step, FeMo-co maturation occurs at NifEN upon incorporation of Mo and homocitrate, which depend on transient NifH-NifEN associations27,28,29,30. The de novo synthesized FeMo-co is transferred to apo-NifDK, converting the inactive protein into active holo-NifDK. In the third step, ARA is often used to assess the level of holo-NifDK activity (and hence FeMo-co synthesis).

Following the FeMo-co synthesis and insertion reactions, tetrathiomolybdate was added to prevent further FeMo-co incorporation into apo-NifDKAv during the acetylene reduction assay. OsNifHHt and ScNifBMt jointly catalyzed the NifB-dependent synthesis of FeMo-co in vitro. FeMo-co synthesis confirmed the compatibility of OsNifHHt and ScNifBMt as demonstrated by the in vitro maturation of NifDK (Fig. 4). It also demonstrated the interspecies compatibility of OsNifHHt, ScNifBMt, NifENAv and NifDKAv, collectively establishing the conserved biochemical core of nitrogenase.

Fig. 4: NifB-dependent in vitro FeMo-co synthesis using OsNifHHt and ScNifBMt.
figure 4

In vitro FeMo-co synthesis was performed by combining M. thermautotrophicus NifB purified from yeast (ScNifBMt) with OsNifHHt (bar 1). The subsequent ARA was performed using a 20:1 molar ratio of NifHAv to NifDKAv. To inhibit apo-NifDKAv activation by FeMo-co produced during the ARA, tetrathiomolybdate was added to all vials following in vitro FeMo-co synthesis and insertion into apo-NifDKAv. Bar 2 represents the background activity observed from using ScNifBMt in the absence of OsNifHHt. As control reactions for the functionality of apo-NifENAv and apo-NifDKAv, FeMo-co synthesis was performed using ScNifHHt in the presence (Pos ctrl) or absence (Neg ctrl) of purified NifB-co. Data are means ± SD, n = 3 (bars 1 and 2) and n = 2 (bars 3 and 4) technical replicates.


Engineering BNF in plants is a major longstanding goal of plant biotechnology. Earlier strategies to reduce global dependence on N fertilizers included the use of diazotrophic bacteria to colonize the rhizosphere31,32,33,34,35,36. The introduction of bacterial nif genes into cereals to increase crop productivity offers a more direct approach in which the plants fix their own N. Multigene transfer technology allows the optimization of different combinations of heterologous genes from diverse origins, as well regulatory elements such as promoters and targeting peptides. However, remaining challenges include the O2 sensitivity of nitrogenase and its accessory proteins, the complexity of the machinery that provides metal clusters to nitrogenase components9, and the intricate regulation of nitrogenase expression and activity37.

Many nif genes are involved in the assembly and activity of nitrogenase and its metal cofactors in bacteria. The essential bacterial genes that must be transferred to cereals because there are no corresponding endogenous proteins include at least nifH, nifD, nifK nifB, nifE and nifN. In contrast, some or all of the accessory genes, such as nifU, nifS, nifQ and nifV (which provide the FeMo-co building blocks: [Fe-S] clusters, molybdenum, and homocitrate), as well as nifJ and nifF (which are involved in electron transfer to NifH) show at least partial functional redundancy with plant proteins7,38. Plant counterparts of the NifU/NifS system are found in the mitochondria, which could therefore provide a ready source of [Fe-S] clusters as well as energy and the near anoxic environment necessary to protect the heterologous nitrogenase39. It is not yet possible to directly transform the plant mitochondrial genome, but proteins encoded by nuclear transgenes can be directed to the mitochondria if provided with the appropriate N-terminal targeting peptide.

The expression of an active Fe protein in plant mitochondria thus requires at least nifH and nifM, and possibly also nifU and nifS. When Klebsiella pneumoniae NifH was produced in E. coli, only NifM was required for NifH functionality16,40, suggesting that [4Fe-4S] cluster insertion is less specific and can be achieved by the host machinery to some extent. In yeast, NifU and NifS were needed for the provision of [Fe-S] clusters to confer NifB activity41, but not for NifH12. This may reflect the distinct mechanisms used to incorporate [Fe-S] clusters into the NifB and NifH proteins, or the different requirements for the clusters. While NifH contains a single permanent [4Fe-4S] cluster only required for catalysis, NifB has three distinct [4Fe-4S] clusters9. One is a SAM-coordinated cluster (the RS-cluster) that is needed for the catalytic activity of NifB, and the other two are [4Fe-4S] clusters (K1 and K2-clusters) that are the substrate clusters for NifB-co formation. The expression of active NifB therefore requires constant biosynthesis and insertion of [4Fe-4S] clusters to replenish those used for NifB-co. The heterologous host also influences the activity of Nif proteins. For example, when NifH was transiently co-expressed with NifM, NifU and NifS in N. benthamiana mitochondria, its activity was lower than in yeast15. In vitro reconstitution restored activity to N. benthamiana NifH, suggesting that the protein isolated from leaf mitochondria was to large extent lacking the required [4Fe-4S] clusters.

The first active Fe protein reported in higher plants was A. vinelandii NifH expressed together with NifM in transplastomic tobacco13. However, active Fe protein was only detected when the plants were incubated in a 10% O2 atmosphere13. The same transplastomic plants were later grown in normal air, and Fe protein activity was detected when leaves were collected at the end of the dark period and when the protein extract was heated23. The heat treatment precipitated apo-NifH protein lacking [4Fe-4S] clusters, and therefore enriched the holo-protein, increasing the specific activity of the remaining NifH protein. The study suggested that the endogenous [Fe-S] cluster biosynthesis system in plastids was, like that in N. benthamiana mitochondria, insufficient for complete NifH maturation23. Similar results were reported by the transient expression of soluble A. vinelandii NifH, NifM, NifU and NifS in N. benthamiana chloroplasts14. NifHAv was active when the leaves were harvested at the end of the dark period, but only when co-expressed with NifUAv and NifSAv, confirming that chloroplast assembly and transfer factors are unable to provide a sufficient quantity of [Fe‐S] clusters for the assembly of a functional NifH14.

After screening 32 diverse NifH proteins in N. benthamiana to compare expression level, solubility, and functionality NifHHt was found to have superior properties for plant expression15. For this reason, here we used the TS-tag and STAC to purify NifHHt from stably transformed rice plants and callus. The OsNifHHt protein migrated as one major band in both rice callus and plants, confirming correct processing in the mitochondria. The isolated OsNifHHt protein was colorless, but this was expected given its low concentration in rice extracts compared to those from bacteria or yeast. Most importantly, the OsNifHHt protein was soluble and stable in rice mitochondria, although not fully equipped with its [4Fe-4S] cluster, and could readily be activated when complemented with [4Fe-4S] cluster-loaded NifU in vitro. This is essential because the Fe protein must be abundant, stable and soluble for successful nitrogenase engineering in plants. The accumulation of an unstable NifH protein could induce cellular stress and damage if exposed to O2, or if [4Fe-4S] clusters were limited for other reasons. The efficient in vitro reconstitution of OsNifHHt protein indicated that future research should focus on better Fe transport, [Fe-S] cluster biosynthesis, delivery to Nif proteins and protection. In this regard, repeated attempts to co-express NifU and NifS from A. vinelandii were unsuccessful in generating detectable protein accumulation in rice callus and regenerated plants despite high levels of mRNA expression (Supplementary Fig. 12). Alternatively, [Fe-S] cluster delivery to Nif proteins could be enhanced by manipulating the host Fe homeostasis (using Fe fortified rice lines) and endogenous mitochondrial pathways.

Our results agree with data previously reported for N. benthamiana leaves (transient expression). The yield of NbNifHHt was ~5–6 mg kg−1 leaf tissue using STAC purification, but the activity of the as-isolated enzyme was low (maximum 25 nmol ethylene min−1 mg−1 when NifH was used at a 40-fold molar ratio to NifDK). However, NifH reconstituted by the in vitro transfer of [4Fe-4S] clusters from NifU increased the activity of NbNifHHt to 250 nmol ethylene min−1 mg−1. Although we purified relatively low amounts of NifHHt from rice plants (0.25–0.5 mg kg−1) compared to N. benthamiana, the yield of rice callus (~6 mg kg−1) was similar to that reported in N. benthamiana and the activity of the protein from rice callus was similar to that of the protein from N. benthamiana leaves. Furthermore, the activity of reconstituted OsNifHHt was ~9-fold higher than the as-isolated enzyme (50 vs 450 nmol ethylene min−1 mg−1) and identical to NifHHt expressed in yeast, which probably reflects its maximum activity in this assay with a full complement of [4Fe-4S]. Importantly, the reconstituted and as-isolated enzymes from yeast showed similar activities (~450 nmol ethylene min−1 mg−1), confirming that NifHHt expressed in yeast mitochondria is provided with sufficient [Fe-S] clusters, which was not the case in rice. To enhance nitrogenase activity, it will therefore be necessary to characterize the insertion of [Fe-S] into NifH in planta and target this process for improvement.

In summary, we have demonstrated the expression of NifHHt and NifMAv at the mRNA and protein levels in stably transformed rice callus and regenerated plants. We generated rice plants targeting nitrogenase Fe protein to the mitochondria. Fe protein purified from rice was stable, soluble, and capable of electron transfer to NifDKAv, confirming the incorporation of endogenous rice mitochondrial [4Fe-4S] clusters, albeit at low levels. The stable accumulation of Fe protein in transgenic rice, a major staple crop, represents a critical step toward the expression of a complete functional Nif complex as required to achieve BNF in cereals, although the levels of active Fe protein obtained in this study are likely not yet sufficient for this goal. Further research should focus on increasing the occupancy and/or stability of NifH [4Fe-4S] clusters, wiring NifH to the cellular electron transfer machinery, and making the overall energetics more favorable for nitrogenase maturation and activity in plant mitochondria.


Genetic constructs

The H. thermophilus nifH and A. vinelandii nifM, nifS, nifU genes were codon optimized for S. cerevisiae using the GeneOptimizer tool (Thermo Fisher Scientific, Waltham, MA, USA) and synthesized by Thermo Fisher Scientific as part of the Engineering Nitrogen Symbiosis for Africa (ENSA) project. The ZmUbi1 + 1sti:cox4-twinstrep-nifHHt:tNos construct was transferred to vector pUC57 (GenScript, Piscataway, NJ, USA) by digesting the empty vector with BamHI and PstI and inserting the synthetic ZmUbi1 + 1sti sequence, then digesting the intermediate vector with PstI and SphI and inserting the synthetic cox4-twinstrep-nifHHt:tNOS sequence. The pOsActin:su9-nifMAv:tNos construct was transferred to pUC57 by digesting the empty vector with ACC65I and XbaI and inserting the OsActin sequence, then digesting the intermediate vector with XbaI and SaII and inserting the synthetic su9-nifMAv:tNos sequence. The ZmUbi1 + 1sti:su9-nifSAv:tADH1 construct was transferred to vector pUC57 by digesting the empty vector with EcoRI and BamHI inserting the synthetic ZmUbi1 + 1sti sequence, then digesting the intermediate vector with BamHI and HindIII and inserting the synthetic su9-nifSAv:tADH1 sequence. The ZmUbi1 + 1sti:su9-nifUAv:tCYC1 construct was transferred to vector pUC57 by digesting the empty vector with EcoRI and SacI and inserting the synthetic ZmUbi1 + 1sti sequence, then digesting the intermediate vector with SacI and BamHI and inserting the synthetic su9-nifUAv:tCYC1 sequence. All restriction enzymes and T4 DNA ligase were obtained from Promega (Madison, WI, USA). Ligated plasmids were introduced into chemically competent E. coli DH5α cells and selected on lysogeny broth (LB) supplemented with 100 µg mL−1 ampicillin. The inserts were confirmed by Sanger sequencing (Stabvida, Caparica, Portugal). The plant expression vectors, and the sequences of the genetic components, are listed in Supplementary Table 1. Primers used for vector construction are listed in Supplementary Table 2.

Transformation of rice explants, callus recovery and regeneration of transgenic plants

Six-day-old mature rice zygotic embryos (Oryza sativa cv. Nipponbare) were isolated from surface-sterilized seeds and transferred to Murashige and Skoog (MS) osmoticum medium prepared using 4.4 g L−1 MS powder (Duchefa Biochemie, Haarlem, Netherlands) supplemented with 0.3 g L−1 casein hydrolysate, 0.5 g L−1 proline, 72.8 g L−1 mannitol, 30 g L−1 sucrose and 2.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) 4 h before bombardment with 10 mg gold particles coated with the nifH and nifM plasmids and the hpt plasmid at a 3:3:1 molar ratio42. The same method was used for co-transformation of nifS and nifU genes, to assess expression. The embryos were returned to osmoticum MS medium for 16 h before selection on standard MS medium (as above without mannitol) supplemented with 2.5 mg L−1 2,4-D and 50 mg L−1 hygromycin in the dark for 2–3 weeks. One half of each callus clone was maintained in an undifferentiated state, and the other half was transferred to regeneration medium. Regenerated plantlets were transferred to soil and grown in large, flooded trays in growth chambers (28/25 °C day/night temperature with a 12-h photoperiod and 80% relative humidity)43. Plants were irrigated with tap water containing 100 μM Fe(III)-EDDHA (Sequestrene 138 Fe G-100; Syngenta Agro, Madrid, Spain). The Fe(III)-EDDHA solution in the trays was replaced every week. T0 plants were grown to an age of 1–2 months and harvested at the end of the dark period by cutting the whole plant down to 2–3 cm above soil level.

Gene expression analysis by quantitative real-time PCR

Total RNA was isolated from rice callus and corresponding regenerated plant leaves using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). First-strand cDNA was synthesized from 1 µg total RNA using Ominiscript Reverse Transcriptase (Qiagen) and quantitative real-time PCR (qRT-PCR) was carried out by CFX96 system (Bio-Rad, Hercules, CA, USA) using a 15 μl mixture containing 1.0 μl 5-fold diluted cDNA template, 2× iTaq SYBR Green Supermix (Bio-Rad) and 0.5 μM of each primer44. The gene-specific primers listed in Supplementary Table 2. The identity of the PCR products was confirmed by sequencing. Expression levels were normalized against OsActin mRNA. Three technical replicates were tested for each sample.

Protein extraction and immunoblot analysis

Soluble rice protein extracts were prepared by grinding ~50 mg leaf tissue (snap-frozen in liquid N2) in 2 mL Eppendorf tubes using 3 mm steel-balls and a BeadBug microtube homogenizer (Benchmark Scientific, Sayreville, NJ, USA) operating at 400 rpm for 20 s. Leaf powder was resuspended in seven volumes (v/w) of extraction buffer comprising 100 mM Tris-HCl (pH 8.6), 200 mM NaCl and 10% glycerol, supplemented with 1 mM PMSF, 1 μg mL−1 leupeptin and 5 mM EDTA. After three rounds of homogenization, cell debris was removed by centrifugation at 20,000 × g for 5 min at 4 °C, and the supernatant was collected and stored at −80 °C.

Rice proteins were separated by SDS-PAGE and then immunoblotted to Protran Premium 0.45 µm nitrocellulose membranes (GE Healthcare, Chicago, IL, USA) using a semidry transfer apparatus (Bio-Rad) at 20 V for 45 min. Similar loading was confirmed by staining polyacrylamide gels with Coomassie Brilliant Blue or staining of nitrocellulose membranes with Ponceau S. The membranes were blocked with 5% non-fat milk in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.02% Tween-20) for 1 h at room temperature before incubation with primary antibodies overnight at 4 °C. Primary monoclonal antibodies specific for strep-tagged OsNifHHt (Strep-MAB, 2-1507-001; IBA Lifesciences, Göttingen, Germany), or polyclonal antibodies specific for NifH or NifM (polyclonal rabbit antibodies generated against A. vinelandii NifH and NifM proteins) or RuBisCO were diluted 1:2000–1:5000 in TBST supplemented with 5% bovine serum albumin (BSA). Secondary antibodies (Invitrogen, Thermo Fisher Scientific) were diluted 1:20,000 in TBST supplemented with 2% non-fat milk and incubated for 2 h at room temperature. Membranes were developed on medical X-ray films (AGFA, Mortsel, Belgium) using enhanced chemiluminescence.

Purification of NifH by STAC

OsNifHHt protein extracts were prepared for STAC purification at O2 levels below 1 ppm in anaerobic chambers (Coy Laboratory Products, Grass Lake, MI, USA, or MBraun, Garching, Germany). Callus (175 g for line HtH189, 234 g for HtH200 and 55 g for HtH202) was snap-frozen in liquid nitrogen, transferred inside the glovebox and then resuspended in lysis buffer comprising 100 mM Tris-HCl (pH 8.5), 200 mM NaCl, 10% glycerol, 3 mM sodium dithionite (DTH), 5 mM 2-mercaptoethanol (2-ME), 1 mM PMSF, 1 μg mL−1 leupeptin, 10 μg mL−1 DNAse I, and 1:200 (v/v) BioLock solution (2-0205-050, IBA Lifesciences) at a ratio of 1:2 (w/v). For purification of OsNifHHt from HtH200 plants, above-ground tissue (45 and 90 g for 1- and 2-months-old plants, respectively) from Fe fertilized plants was harvested at the end of the 12 h dark cycle (before the onset of light), snap-frozen in liquid nitrogen, transferred inside the glovebox and then resuspended in the lysis buffer at a ratio of 1:4 (w/v). The amounts of callus and plant tissue used for purifications are reported as fresh-weight. Callus and plant total extracts were prepared by mechanical disruption under an anaerobic atmosphere using a blender (Oster 4655) modified with a water-cooling system operating at full speed in four cycles of 2 min at 4 °C. The total extract was transferred to centrifuge tubes equipped with sealing closures (Beckman Coulter, Brea, CA, USA) and centrifuged at 50,000 × g for 1.5 h at 4 °C using an Avanti J-26 XP device (Beckman Coulter). The supernatant was passed through filtering cups with a pore size of 0.2 μm (Nalgene, Thermo Fisher Scientific) to produce a cell-free extract containing soluble proteins. This was loaded at a flow rate of 2.5 mL min−1 onto a 5 mL Strep-Tactin XP column (IBA LifeSciences) attached to an ÄKTA FPLC system (GE Healthcare). The column was washed at 16 °C with 150 mL washing buffer comprising 100 mM Tris-HCl (pH 8.0), 200 mM NaCl, 10% glycerol, 2 mM DTH and 5 mM 2-ME. Bound proteins were eluted with 15–20 mL washing buffer supplemented with 50 mM biotin (IBA LifeSciences). The elution fraction was concentrated using an Amicon Ultra centrifugal filter with a cut-off pore size of 10 kDa (Merck Millipore, Burlington, MA, USA). Biotin was removed by passing the protein through PD-10 desalting columns (GE Healthcare) equilibrated with washing buffer. Desalted eluate was further concentrated and snap-frozen in cryovials (Nalgene) and stored in liquid nitrogen.

Quantification of purified OsNifHHt protein, N-terminal sequencing and MS analysis

The purified OsNifHHt protein was quantified by the densitometric analysis of Coomassie-stained gels using ImageJ45 compared to a standard of yeast ScNifBMi protein as shown in Supplementary Fig. 4. N-terminal amino acid sequences were determined by Edman degradation (Centro de Investigaciones Biológicas, Madrid, Spain). OsNifHHt protein (~250 pmol) was separated by SDS-PAGE, transferred to a Sequi-Blot PVDF membrane with a 0.2 µm pore size (Thermo Fisher Scientific) in 50 mM borate buffer (pH 9.0), stained with freshly prepared Coomassie R-250 (Sigma-Aldrich, St Louis, MO, USA) (0.1% in 40% methanol and 10% acetic acid), and then destained using 50% methanol. OsNifHHt protein (~60 pmol) was separated by SDS-PAGE followed by Coomassie staining and destaining as described above. Protein excised from the gel was analyzed by mass spectrometry at the Universidad Complutense de Madrid, Spain.

In vitro NifH activity analysis

OsNifHHt activity was determined in an anaerobic chamber as described15. Purified OsNifHHt was analyzed using an acetylene reduction assay following the addition of NifDKAv and ATP-regenerating mixture (1.23 mM ATP, 18 mM phosphocreatine, 2.2 mM MgCl2, 3 mM DTH, 46 μg mL−1 creatine phosphokinase, 22 mM Tris-HCl pH 7.5) in a final volume of 400 μL in 9 mL serum vials containing 500 μL acetylene under an argon atmosphere. The assay was performed at 30 °C in a shaking water bath for 20 min. Reactions were stopped by adding 100 μL 8 M NaOH. Positive control reactions for acetylene reduction were carried out with NifHAv. The ethylene formed in the reaction was measured in 50-μL gas-phase samples using a Porapak N 80/100 column in a gas chromatograph (Shimadzu, Duisburg, Germany). The ethylene peak from reactions devoid of both NifH and NifDK proteins (no nitrogenase component added) in each experiment was subtracted from the corresponding reported ARA activities.

In vitro [Fe-S] cluster reconstitution and reconstituted NifH activity

OsNifHHt [Fe-S] cluster reconstitution in anaerobic chambers was carried out by incubating with EcNifUAv (A. vinelandii NifU expressed in and purified from E. coli cells) previously loaded with [4Fe-4S] clusters. First, 20 μM NifU dimer was added to 100 mM MOPS (pH 7.5) supplemented with 8 mM 1,4-dithiothreitol (DTT) and the reaction was incubated at 37 °C for 30 min. We then added 1 mM l-cysteine, 1 mM DTT, 300 μM (NH4)2Fe(SO4)2, and 225 nM NifSAv purified from E. coli (EcNifSAv) to the reduced NifU and incubated the reaction on ice for 3 h. Finally, the proteins were diluted 40,000-fold in 100 mM MOPS (pH 7.5) and concentrated using Amicon centrifugal filters with a 30-kDa cut off to remove excess reagents. Isolated OsNifHHt protein was mixed with the [4Fe-4S] cluster-reconstituted EcNifUAv (NifU-mediated reconstitution) and then immediately used for the acetylene reduction assay.

In vitro FeMo-co synthesis and apo-NifDKAv reconstitution

NifB-dependent FeMo-co synthesis assays were prepared in anaerobic chambers15. We assembled 100 μL reactions containing 3 μM OsNifHHt, 1 μM ScNifBMt, 9 μM [4Fe-4S] cluster-loaded EcNifUAv, 1.5 μM apo-NifENAv, 0.3 μM apo-NifDKAv, 125 μM SAM, 17.5 μM Na2MoO4, 175 μM R-homocitrate, 1 mg mL−1 BSA, and ATP-regenerating mixture (1.23 mM ATP, 18 mM phosphocreatine, 2.2 mM MgCl2, 3 mM DTH, 46 μg mL−1 creatine phosphokinase, 100 mM MOPS pH 7.5) at 30 °C for 1 h. In the positive control reaction, the assay was carried out by using ScNifHHt instead of the rice purified variant, and NifB-co (20.4 μM Fe) instead of ScNifBMt and EcNifUAv. In the negative control reaction, the reaction mixture contained all the components except for any source of NifB-co or ScNifBMt and EcNifUAv. Following the in vitro synthesis and insertion of FeMo-co, 17.5 μM (NH4)2MoS4 was added to prevent further FeMo-co incorporation into apo-NifDKAv, and the reaction was incubated for 20 min at 30 °C, shaking at 600 rpm. Activation of apo-NifDKAv was analyzed by adding 500 μL ATP-regenerating mixture and NifHAv (2.0 μM final concentration) in 9-mL vials containing 500 μL acetylene under argon gas. The acetylene reduction assay was performed at 30 °C for 20 min, and the resulting ethylene was measured in 50 μL gas-phase samples using a Porapak N 80/100 column as described above.

Statistics and reproducibility

For enzymatic activity assays in plants the standard deviation (SD) of the mean was calculated based on two biological replicates (two technical replicates each). The standard deviation (SD) of the mean for callus activity assays was calculated based on three biological replicates (three technical replicates each). For relative gene expression analysis standard deviations (SD) were calculated based on three technical replicates.