Fig 1.
Transgenes nifH and nifM are integrated into the plastid genome, and transplastomic lines are homoplastomic.
Restriction sites discussed in the manuscript are shown. (A) nif expression cassette from pMON261406. The vector contains a chimeric two-gene nifH and nifM operon expression cassette (Prrn:G10L:nifH:Trps16:G10L:nifM:TpetD). This two-gene operon cassette is cloned upstream of the selectable aadA gene cassette in pMON253685 (Fig 1B) to create plasmid pMON261406 (Fig 1C). (B) pMON253685 contains the the aadA gene expression cassette (PpsbA:aadA:TpsbA). This expression cassette is flanked by regions of homology to the endogenous plastid rbcL gene region (SacI-SbfI fragment) and to the endogenous plastid accD region (PmeI-SphI fragment). The vector pMON253685 is similar to pMON261406 except it lacks the chimeric nifH and nifM expression cassette. (C)The structure of the transplastomic lines NT_S21023687 and NT_S21023688 (produced as a result of the plastid transformation with pMON261406). Restriction enzyme sites used for Southern blot analysis (PvuII and EcoO109I) are located inside and outside of transforming DNA, which is delineated by SacI and SphI restriction enzyme sites. Black boxes represent hybridizing regions of the transplastomic genome; solid lines represent DNA fragments expected to hybridize with gene specific probes (not to scale). Indicated on the right are corresponding sizes (in kb) of the expected DNA fragments from Southern blot analysis that are shown in Figs D-E. DNA fragments and hybridizing regions corresponding to gemonic DNA of wild-type plants (6.0 kb) and the negative control event NT_S21023684 (7.2 kb) are also shown (D-F) Southern blot analyses of wild type and transplastomic lines. Tobacco genomic DNA was extracted from the wild type and transplastomic plants and digested with PvuII or EcoO109I restriction endonuclease. The DNA were separated on an agarose gel, transferred to a nylon membrane and hybridized with a digoxigenin-labeled probe specific to the rbcL gene (that is located close to the expected insertion site of the transgenes), nifH or nifM genes as indicated. Digoxigenin-labeled MWMVII and MWMII (Roche) indicate molecular weight markers, and sizes (kb) of MWM bands are indicated. The NT_S21023684 line (derived from the plastid transformation of pMON253685 and lacking nif genes) was included in the analysis as appropriate. (D) Southern blot of wild-type and transplastomic lines (R0 and R1 generations) after PvuII restriction enzyme digestion and hybridization with the resident rbcL coding region probe. Absence of wild type bands in the transplastomic samples indicates that these lines are homoplasmic for the inserted transgenic cassettes. (E) Southern blot of wild type and transplastomic lines after EcoO109I restriction enzyme digestion and hybridization with the nifH coding region probe. (F) Southern blot of wild type and transplastomic lines after EcoO109I restriction enzyme digestion and hybridization with the nifM coding region probe. For Figs E and F, note the expected absence of hybridizing bands in wild type samples.
Fig 2.
NifH and NifM proteins are expressed in transplastomic lines NT_S21023687 and NT_S2103688, but not in the control line NT_S21023684.
Total protein extracts were analyzed by western blot analysis to detect proteins that were immunoreactive to antibodies raised against either the NifH (31 kDa) or NifM proteins (33 kDa). Lane 1, NT_S21023684 (negative control); lane 2, NT_S21023687; lane 3, NT_S21023688. A weak non-specific band (22 kDa) was detected with antibodies raised against NifM protein in all total protein extracts analyzed in this experiment. Select bands of molecular weight marker (Precision Plus Protein Dual Color Standards, Bio-Rad) are shown.
Fig 3.
Transplastomic plants expressing NifH and NifM proteins produce active Fe subunit of nitrogenase.
Fe-protein enriched fractions from transplastomic lines and corresponding fractions from control plants were combined with bacteria-produced MoFe subunit. The enzymatic activity was evaluated via the acetylene reduction assay (see Materials section). Relative ethylene amounts produced after 3 hour incubations are shown. The experiment was repeated three times using three independent biological replicates (results for which are shown as Replicates 1–3), and error bars represent standard deviation of these three experiments done on three different days. Because ethylene is a minor contaminant in acetylene preparations [45], ethylene level present in the negative control samples (NT_S21023684 line derived from the transformation of pMON253685 which lacked nif genes) was used as the background level and is indicated with a dashed line.