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Plant genotype and rhizobia strain combinations strongly influence the transcriptome under heavy metal stress conditions in Medicago truncatula

Heavy metals such as cadmium (Cd) and mercury (Hg) pose significant threats to plant health and food safety as they are absorbed from the environment. Legumes are generally considered sensitive to heavy metals but possess standing genetic variation for accumulation and tolerance to toxic ions. We conducted a transcriptomic analysis on hydroponically and soil grown Medicago truncatula plants to investigate gene expression responses to Cd and Hg exposure in roots, leaves, and nodules. By using plant genotypes with varying metal tolerance or accumulation levels, we observed distinct clustering of gene ontologies, indicating tissue-specific, genotype-specific, and metal-specific gene expression patterns. Considering the symbiotic relationship between legumes and nitrogen-fixing bacteria, we further examined plant phenotypes and transcriptomes of plant genotypes with contrasting Hg accumulation levels and inoculated them with high or low Hg-tolerant Sinorhizobium medicae strains that have presence-absence variation for a mercury reductase (Mer) operon. Host plants inoculated with the Hg-tolerant rhizobia strain possessing a Mer operon exhibited less reduction in nodule number and plant biomass. A smaller reduction in iron (Fe) distribution in nodules after Hg stress was measured using X-ray Fluorescence (XRF) imaging. Dual transcriptome (host plant and bacteria) analysis of nodules revealed a remarkable decrease in the number of differentially expressed genes (DEGs) and clustering of gene ontologies in plants inoculated with the Hg-tolerant rhizobia strain, including symbiosis related genes. This finding suggests that the Hg-tolerant rhizobia strain has the potential to mitigate Hg stress in host plants. Furthermore, we observed genotype by-genotype interactions between the high Hg accumulating plant genotype and the Hg-tolerant rhizobia strain. These findings provide insights into enhancing plant resilience in contaminated environments through optimizing legume-rhizobia interactions for heavy metal tolerance.

59 BASIC BIOLOGICAL SCIENCES↗

Investigating biological nitrogen fixation via single-cell transcriptomics

The extensive use of nitrogen fertilizers has detrimental environmental consequences, and it is essential for society to explore sustainable alternatives. One promising avenue is engineering root nodule symbiosis, a naturally occurring process in certain plant species within the nitrogen-fixing clade, into non-leguminous crops. Advancements in single-cell transcriptomics provide unprecedented opportunities to dissect the molecular mechanisms underlying root nodule symbiosis at the cellular level. This review summarizes key findings from single-cell studies in Medicago truncatula, Lotus japonicus, and Glycine max. We highlight how these studies address fundamental questions about the development of root nodule symbiosis, including the following findings: (i) single-cell transcriptomics has revealed a conserved transcriptional program in root hair and cortical cells during rhizobial infection, suggesting a common infection pathway across legume species; (ii) characterization of determinate and indeterminate nodules using single-cell technologies supports the compartmentalization of nitrogen fixation, assimilation, and transport into distinct cell populations; (iii) single-cell transcriptomics data have enabled the identification of novel root nodule symbiosis genes and provided new approaches for prioritizing candidate genes for functional characterization; and (iv) trajectory inference and RNA velocity analyses of single-cell transcriptomics data have allowed the reconstruction of cellular lineages and dynamic transcriptional states during root nodule symbiosis.

Lotus japonicus↗

Convergent evolution of NFP -facilitated root nodule symbiosis

The origin and phylogenetic distribution of symbiotic associations between nodulating angiosperms and nitrogen-fixing bacteria have long intrigued biologists. Recent comparative evolutionary analyses have yielded alternative hypotheses: a multistep pathway of independent gains and losses of root nodule symbiosis vs. a single gain followed by numerous losses. A detailed reconstruction of the history of genes involved in signaling between nitrogen-fixing bacteria and potential hosts, particularly lipo-chitooligosaccharide (LCO) signaling, is needed to distinguish between these hypotheses. LCO recognition by plants involves the Nod Factor Perception ( NFP ) gene family; in the legume model Medicago truncatula (Fabales), MtNFP is essential for establishing rhizobial symbiosis. Here, we document convergent evolution of NFP , indicating multiple origins of LCO-driven symbiosis. In contrast to previous models that explain the recruitment of NFP via a single duplication in the ancestor of the nitrogen-fixing clade, our phylogenomic and synteny results suggest this duplication does not span the entire clade. Tandem duplication in a common ancestor of Cucurbitales and Rosales resulted in the NFP1 and NFP2 groups. In contrast, the phylogenetically closest paralog of MtNFP is MtLYR1 , located on a different chromosome within a large syntenic block. All available data indicate that a large-scale duplication resulted in MtNFP and MtLYR1 , likely corresponding to a whole-genome duplication in an ancestor of subfamily Papilionoideae of Fabaceae. We show that MtNFP and the NFP2 -like group are not orthologous, indicating multiple independent gains of NFP -based LCO signaling. This molecular convergence provides a possible mechanism for multiple gains of root nodule symbiosis across the nitrogen-fixing clade.

LCO signaling↗

Major facilitator family transporters specifically enhance caffeyl alcohol uptake during C‐lignin biosynthesis

The mode of transport of lignin monomers to the sites of polymerization in the apoplast remains controversial. C-Lignin is a recently discovered form of lignin found in some seed coats that is composed exclusively of units derived from caffeyl alcohol. RNA-seq and proteome analyses identified a number of transporters co-expressed with C-lignin deposition in the seed coat of Cleome hassleriana. Cloning and influx/efflux analysis assays in yeast identified two low-affinity transporters, ChPLT3 and ChSUC1, that were active with caffeyl alcohol but not with the classical monolignols p-coumaryl, coniferyl, and sinapyl alcohols, consistent with molecular modeling and docking studies. Expression of ChPLT3 in Arabidopsis seedlings enhanced root growth in the presence of caffeyl alcohol, and expression of ChPLT3 and ChSUC1 correlated with lignin C-unit content in hairy roots of Medicago truncatula. We present a model, consistent with phylogenetic and evolutionary considerations, whereby passive caffeyl alcohol transport may be supplemented by hitchhiking on secondary active transporters to ensure the synthesis of C-lignin, and inhibition of synthesis of G-lignin, in the apoplast.

59 BASIC BIOLOGICAL SCIENCES↗

Phylogenomic discovery and engineering of nitrogen fixation into the bioenergy woody crop poplar

Biological nitrogen fixation (BNF) is a key process enabling plants in specific lineages to convert atmospheric dinitrogen (N₂) into bioavailable ammonia through symbioses with diazotrophic microbes. Expanding this capability beyond native nitrogen-fixing clades into non-nodulating crops would reduce synthetic fertilizer use, lowering energy inputs and environmental impacts in agriculture. Supported by DOE Funding Award DE-SC0018247, the NitFix project advanced foundational knowledge required to engineer root-nodule symbioses in new host species. The team generated the most comprehensive phylogenomic analysis to date of all known nodulating lineages, resolving the evolutionary history of nitrogen-fixing symbiosis and identifying core gene suites retained across nodulating taxa. Through multimodal genomics, transcriptomics, and functional analyses in Medicago truncatula and related species, the project mapped regulatory networks underlying nodule organogenesis, bacterial infection, and nitrogen-fixation efficiency. Key discoveries include the identification of conserved signaling modules for rhizobial recognition, transcription factors controlling nodule differentiation, and metabolic pathways integrating fixed nitrogen into plant growth. The project also developed enabling tools—including optimized transformation pipelines, gene-editing workflows, and imaging-based phenotyping—to accelerate engineering efforts in emerging models. Together, these results refine the mechanistic framework of symbiotic nitrogen fixation and highlight transferable components essential for rewiring these traits into non-nodulating crops.

59 BASIC BIOLOGICAL SCIENCES↗