Expression of nitrogenase polypeptides in plant cells

A process for producing a plant cell comprising mitochondria and polynucleotides that encode at least 3 Nif polypeptides selected from the group consisting of NifF, NifW, NifY, NifZ, NifV and a NifD-linker-NifK fusion polypeptide, wherein the at least 3 Nif polypeptides comprise a NifV polypeptide, comprising steps of:

• (i) introducing, into a plant cell, exogenous polynucleotides that encode at least 3 Nif polypeptides selected from the group consisting of NifF, NifW, NifY, NifZ, NifV and a NifD-linker-NifK fusion polypeptide, wherein the at least 3 Nif polypeptides comprise a NifV polypeptide, wherein the exogenous polynucleotides each comprise a promoter which is operably linked to a nucleotide sequence which encodes one of the Nif polypeptides and which expresses the nucleotide sequence in the plant cell, wherein the Nif polypeptides each independently comprise a mitochondrial targeting peptide (MTP), wherein each MTP is at the N-terminus of the Nif polypeptide, wherein the NifD-linker-NifK fusion polypeptide comprises
• (a) a mitochondrial targeting peptide (MTP) which has a C-terminus,
• (b) a NifD polypeptide (ND) which has a N-terminus and a C-terminus,
• (c) an oligopeptide linker, and
• (d) a NifK polypeptide (NK) which has a N-terminus,
• wherein the C-terminus of the MTP of (a) is translationally fused to the N-terminus of the ND such that the MTP and the ND are covalently joined by a peptide bond, and
• wherein the linker is translationally fused to the C-terminus of the ND and the N-terminus of the NK such that the linker is covalently joined to the C-terminus of the ND by a peptide bond and to the N-terminus of the NK by a peptide bond,
• (ii) culturing the plant cell for a sufficient time for the polynucleotides to be expressed;
• (iii) assaying the solubility of the Nif polypeptides, or cleaved products thereof, in the cell or a progeny cell thereof, and
• (iv) selecting a plant cell wherein the at least 3 Nif polypeptides, or cleaved products thereof, are at least partially soluble in the mitochondria.