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PMID:28316608
| Citation |
Allen, RS, Tilbrook, K, Warden, AC, Campbell, PC, Rolland, V, Singh, SP and Wood, CC (2017) Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix. Front Plant Sci 8:287 |
|---|---|
| Abstract |
The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia. |
| Links |
PubMed PMC5334340 Online version:10.3389/fpls.2017.00287 |
| Keywords |
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Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifF from A. tumefaciens, were expressed in the mitochondrial matrix of N. benthamiana. Flavodoxin (NifF) |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifJ from A. tumefaciens, were expressed in the mitochondrial matrix of N. benthamiana. Pyruvate-flavodoxin oxidoreductase (NifJ) |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifB from A. tumefaciens, were expressed in the the mitochondrial matrix of N. benthamiana. FeMo cofactor biosynthesis protein (NifB) |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifQ from A. tumefaciens, were expressed in the mitochondrial matrix of N. benthamiana. Protein NifQ |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifV from A. tumefaciens, were expressed in the mitochondrial matrix of N. benthamiana. (Homocitrate synthase NifV) |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifZ from A. tumefaciens, were expressed in the mitochondrial matrix of N. benthamiana. (Protein NifZ) |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifM from A. tumefaciens, were expressed in the mitochondrial matrix of N. benthamiana. Putative peptidyl-prolyl cis-trans isomerase NifM |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifH from A. tumefaciens, were expressed in the mitochondrial matrix of N. benthamiana. Nitrogenase iron protein (NifH) |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifK from A. tumefaciens, were expressed in the the mitochondrial matrix of N. benthamiana. Nitrogenase molybdenum-iron protein beta chain (NifK) |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifE from A. tumefaciens, were expressed in the mitochondrial matrix of N. benthamiana. Nitrogenase iron-molybdenum cofactor biosynthesis protein (NifE) |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifS from A. tumefaciens, were expressed in the mitochondrial matrix of N. benthamiana. Cysteine desulfurase (NifS) |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifY from A. tumefaciens, were expressed in the mitochondrial matrix of N. benthamiana. Protein NifY |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifN from A. tumefaciens, were expressed in the mitochondrial matrix of N. benthamiana. Nitrogenase iron-molybdenum cofactor biosynthesis protein NifN |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifU from A. tumefaciens, were expressed in the mitochondrial matrix of N. benthamiana. (Nitrogen fixation protein NifU) |
complete | ||||
| GO:0033650: host cell mitochondrion |
ECO:0000314: |
C |
NifX from A. tumefaciens, were expressed in the mitochondrial matrix of N. benthamiana. (Protein NifX) |
complete | ||||
Notes
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