• Okada, Shoko
• Gillespie, Vanessa
• Johnston, Ema
• Hussain, Dawar
• Wood, Craig

Abstract

Industrial fertiliser is intrinsic to modern agriculture but is expensive and environmentally damaging. To reduce its usage, we are aiming to reconstitute bacterial nitrogenase in plant mitochondria, which requires expression of soluble, functional Nif proteins in the organelle. Here we present our testing pipeline to assess processing, abundance, solubility and function of 16 Klebsiella oxytoca Nif proteins targeted to the mitochondrial matrix using a 51 amino acid MTP, pFAγ51. We found that despite use of the same constitutive promoter and mitochondrial targeting peptide (MTP), MTP::Nif protein abundance as well as processing varied considerably. We then assessed the functional consequence of the N-terminal modifications required for mitochondrial targeting using a bacterial assay. Although most scar9::Nif proteins tolerated the extension, for scar9::NifM the activity was reduced to ~10%. Using proteomics, we detected a ~10-fold increase in scar9::NifM protein expression, and this dysregulation may account for the reduction seen in nitrogenase activity. Finally, we assessed solubility of all pFAγ51::Nif proteins extracted from plants and found activity NifF, M, N, S, U, W, X, Y and Z were soluble, although NifB, E, H, J, K, Q and V were mostly insoluble. Based on mitochondrial processing and solubility, and retention of function in a bacterial assay we identified that NifF, N, S, U, W, Y and Z could be considered suitable for expression in plant mitochondria.Future work can now focus on improving the remaining components to assemble a complete set of plant-ready Nif proteins for reconstituting function in plant mitochondria.

Topics

• impedance spectroscopy