Blasio, F. | bioRxiv Science

bioRxiv2026

Natural variation in NifU and NifS enhances chloroplasts compatibility for nitrogenase engineering

Reconstitution of functional nitrogenase in plants requires the coordinated expression of the [Fe-S] cluster assembly proteins NifU and NifS. However, the extent to which these proteins interact with endogenous Fe-S metabolism and affect plant physiology remains unclear. Here, we compared NifU and NifS homologs from diverse diazotrophs to identify variants compatible with the plant chloroplast environment. Selected variants of Azotobacter vinelandii, Fischerella thermalis, and Marinobacter lutimaris were characterized by transient expression in Nicotiana benthamiana and stable transformation in rice. Plant-produced NifU was largely devoid of [Fe-S] clusters when isolated but retained strong capacity for in vitro [Fe-S] cluster reconstitution and apo-NifH activation in a Ft > Av >Ml gradient, indicating correct folding and function but limited cluster loading or stability in vivo. NifU and NifS expression in transgenic rice resulted in variant-dependent proteome and phenotype effects, with A. vinelandii-expressing lines exhibiting severe defects, F. thermalis lines showing intermediate phenotype, and M. lutimaris lines being indistinguishable from wild type. These results reveal a trade-off between the biochemical activity of NifU and NifS and their compatibility with host metabolism, which must be considered for successful nitrogenase engineering in plants. HighlightNifU/NifS homolog selection determines trade-offs between [Fe-S] cluster assembly activity and plant compatibility, identifying variants that minimize physiological disruption while supporting nitrogenase cofactor assembly in chloroplasts.

plant biology↗

bioRxiv2025

Root growth promotion by Penicillium melinii: mechanistic insights and agricultural applications

O_LIThis study characterizes Penicillium melinii, an endophytic fungus isolated from Arabidopsis thaliana roots, as a plant growth-promoting fungus with potential use as a model to study root development and as a biostimulant for sustainable agriculture. Although endophytes are known to promote plant growth, the underlying molecular mechanisms often remain poorly understood. Here, we aimed to elucidate how P. melinii enhances root system development and to assess its applicability across different crops. C_LIO_LIPhenotypic assays were conducted in Arabidopsis, quinoa and tomato under in vitro, greenhouse and field conditions. Root architecture and biomass were quantified using image-based phenotyping. Transcriptomic and phytohormone profiling assessed plant responses, and fungal genome sequencing coupled with secretome analysis was used to identify candidate effectors and metabolic traits. C_LIO_LIP. melinii consistently promoted root growth and increased plant biomass across species and environments, both in vitro and in the greenhouse. In tomato field trials, this translated into a significant increase in yield. The fungus colonized root surfaces without vascular penetration and triggered a mild transcriptomic response: early activation of stress-response genes followed by their attenuation and sustained upregulation of auxin-related pathways. Notably, the interaction modulates the SLR-ARF-LBD pathway and the number of pre-branch sites probably through increased auxin signalling in the oscillation zone. Additional hormonal changes were limited and mainly associated with the attenuation of the plant response to microorganisms. C_LIO_LIP. melinii enhances lateral root formation through a subtle molecular and metabolic dialogue with the host plant, underscoring its relevance as a model for studying root developmental plasticity. Its strong and reproducible growth-promoting effect, demonstrated with different fungal strains and under controlled and field conditions, supports its potential as a biostimulant for sustainable crop production. C_LI

plant biology↗