The González-Guerrero Lab
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Progress and perspectives in nitrogenase engineering in plants and yeast
Abstract
Summary
Crop productivity relies on synthetic nitrogen fertilizers, which entail significant economic and environmental costs. Engineering crop plants to produce nitrogenase allowing them to acquire nitrogen from the atmosphere offers a transformative approach to sustainable agriculture. However, engineering the nitrogenase pathway into eukaryotic cells poses significant challenges due to the enzyme metal clusters, high energy demands, and extreme sensitivity to oxygen. This review summarizes recent advances in nitrogenase engineering in yeast and plants. Progress has been made using a stepwise modular approach and a functional validation pipeline for core and ancillary nitrogenase proteins. Key milestones include the production of active NifH and NifB proteins in mitochondria and chloroplasts, the biosynthesis of nitrogenase FeMo-co using proteins synthesized in mitochondria, and the expression of functional nitrogenase components in transgenic rice. We discuss the importance of organelle targeting, the exploitation of natural diversity and engineered proteins, and synthetic biology tools for the assembly of multigene pathways. Finally, we present the main remaining obstacles, such as the assembly of functional NifDK in plants, integration with host metabolism and coordinated regulation, and we outline the prospects for the development of nitrogen-fixing crops.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Molecular dissection of zinc-mediated immunity in Arabidopsis thaliana.
Abstract
Summary
Zinc is an essential micronutrient at low concentrations, yet it becomes toxic at slightly higher ones. This is exploited by plants as an effective defensive strategy. However, the molecular components that are involved zinc-mediated immunity remain poorly defined. Here, we show that mixed-linked β-1,3/1,4-glucans naturally ocurring in microbial and grass cell walls and used as an agrobiological solution, trigger zinc accumulation in the Arabidopsis apoplast and upregulate the expression of the zinc transporters HMA2 and HMA4. This response occurs independently of salicylic acid, jasmonic acid and ethylene-mediated signalling pathways, but it requires the LysM receptor kinases CERK1, LYK4 and LYK5, indicating a specific pattern triggered immunity-associated mechanism. We further demonstrate that hma2hma4 mutants display constitutive activation of a broad set of defence-related genes, yet this transcriptional reprogramming is insufficient to confer resistance against the necrotrophic fungus Plectosphaerella cucumerina BMM. Moreover, metabolomic profiling highlights the contribution of specialized metabolites to this defective defence output. Altogether, our findings reveal that zinc-mediated toxicity constitutes a defence mechanism integrated into the immune response triggered by specific microbial or damage associated molecular patterns.
Funder Information Declared
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- Ministerio de Ciencia, Innovación y Universidades, TED2021-131769B-I00, SEV-2016-0672, CEX2020-000999-S, RYC2022-037020-I, CEX2019-000902-S
- Generalitat de Catalunya, CERCA Program
- European Union, HORIZON-MSCA-2022-PF-01-101104098-ZINMUNITY
- Deutsches Elektronen-Synchrotron DESY, I-20230698, I-20250029
- Paul Scherrer Institute, 20221807
Medicago truncatula Iron-chaperone 1 (ICHAP1) is required for symbiotic nitrogen fixation
Abstract
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.
Funder Information Declared
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- Ministerio de Ciencia, Innovación y Universidades
- U.S. National Science Foundation, https://ror.org/021nxhr62, DBI-0703285, IOS-1127155, DBI-2233714
Enhances production of nitrogenase components in Nicotiana benthamiana through co-expression with Bacterioferritin A.
Abstract
Summary
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Engineering 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
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To 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.
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BfrA 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.
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Our 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.
Funder Information Declared
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- Agencia Estatal de Investigación, ID2021-12460OB-100, PCI2021-122052-2B, FJCI-2017-33222, PRE2022-101253
- Instituto de Salud Carlos III, PT17/0019
- Gates Foundation, INV-005889, INV-067006