Biology subjects

Rodriguez-Simon, M.

Publications and source records attributed to Rodriguez-Simon, M..

3 recordsLinked to original sources

bioRxiv2026

Enhanced production of nitrogenase components in Nicotiana benthamiana through co-expression with Bacterioferritin A

O_LIEngineering nitrogen fixing crops requires not only transferring the nitrogenase structural genes, but also the accessory genes to synthesize its iron-sulphur cofactors. Scaffold protein NifU is a critical element in this system as the starting point of nitrogenase cofactor assembly. NifU has been successfully produced in plants, however, its optimal production required high levels of iron in the medium. This is likely due to a faulty connection with the endogenous iron trafficking network C_LIO_LITo identify specific elements targeting iron to NifU, pull-down assays were performed to identify showing bacterioferritin A (BfrA) as a likely candidate. Co-immunopurification, mutant characterization, iron transfer assays, and co-expression in Nicotiana benthamiana assays were carried out. C_LIO_LIBfrA transfers iron to NifU through protein-protein interactions. When these two proteins were co-expressed in N. benthamiana leaves, there was an increase in NifU production. In turn, it led to doubling NifH synthesis, a nitrogenase structural protein that is also required for the synthesis of the more complex nitrogenase cofactors. C_LIO_LIOur results provide a new element towards engineering nitrogen-fixing crops. They also underscore the importance of transferring the metal delivery systems when expressing metalloproteins in heterologous systems. C_LI

Plant Biology↗

bioRxiv2026

Medicago truncatula Iron-chaperone 1 (ICHAP1) is required for symbiotic nitrogen fixation

Hundreds of proteins in the cell require iron (Fe) or Fe-containing cofactors to function. However, how Fe2+ or Fe3+ are specifically allocated to each of these proteins in plant cells remains largely unknown. It has been proposed that Fe metalation could be driven by specific interactions with Fe-shuttling proteins known as Fe-chaperones. Here, we present the first family of plant Fe2+-chaperones (ICHAPs) with orthologues in dicots and monocots. The role of these proteins in Fe distribution to Fe-dependent metabolic processes has been illustrated using symbiotic nitrogen fixation in Medicago truncatula root nodules. ICHAP1 is a soluble Fe2+-binding protein that interacts with plasma membrane Fe2+ transporter NRAMP1, but not with symbiosome Fe2+-transporters. ICHAP1 mutants present altered Fe distribution in cells and they cannot fix nitrogen. A second family member, ICHAP2 is required to target Fe2+ to symbiosomes, as it accepts Fe2+ from ICHAP1 and interacts with symbiosome Fe2+-importer VTL8, but not with NRAMP1. These results indicate a path for Fe2+ allocation from the plasma membrane to the symbiosome through specific protein-protein interactions and Fe2+ exchange from NRAMP1 to ICHAP1, to ICHAP2, and to VTL8.

plant biology↗

bioRxiv2023

Nodule-specific Cu+-chaperone NCC1 is required for symbiotic nitrogen fixation in Medicago truncatula root nodules

Cu+-chaperones are a diverse group of proteins that allocate Cu+ ions to specific copper-proteins, creating different copper pools targeted to specific physiological processes. Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper e.g. for energy delivery via respiration, for which targeted copper deliver systems would be required. MtNCC1 is a nodule-specific Cu+-chaperone encoded in the Medicago truncatula genome, with a N-terminus Atx1-like domain that can bind Cu+ with picomolar affinities. This gene is expressed primarily from the late infection zone to the early fixation zone, and is located in the cytosol, associated to plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation, ncc1 mutants have a severe reduction of nitrogenase activity, and a 50% reduction in copper-dependent cytochrome c oxidase activity. A subset of the copper-proteome is also affected in the mutant nodules. Many of these proteins can be pulled-down when using a Cu+-loaded N-terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper-dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.

plant biology↗