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All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

A substrate-multiplexed platform for profiling enzymatic potential of plant family 1 glycosyltransferases

Plants have expanded various biosynthetic enzyme families to produce a wide diversity of natural products; however, most enzymes encoded in plant genomes remain uncharacterized, highlighting the need for new functional genomic approaches. Here, we report a platform enabling the rapid functional characterization of plant family 1 glycosyltransferases, which serve important roles in plant development, defense, and communication. Using substrate-multiplexed reactions, mass spectrometry, and automated analysis, we screen 85 enzymes against a diverse library of 453 natural products, for a total of nearly 40,000 possible reactions. The resulting dataset reveals a widespread promiscuity and a strong preference for planar, hydroxylated aromatic substrates among family 1 glycosyltransferases. We also characterize glycosyltransferases with an unusually wide substrate scope and with a non-canonical Cys-Asp catalytic dyad. This work establishes a widely-applicable enzymatic screening pipeline, reflects the immense glycosylation capability of plants, and has implications in biocatalysis, metabolic engineering, and gene discovery.

Sirirungruang, Sasilada↗
DOE OSTI2023

Validation of a metabolite–GWAS network for Populus trichocarpa family 1 UDP-glycosyltransferases

Metabolite genome-wide association studies (mGWASs) are increasingly used to discover the genetic basis of target phenotypes in plants such as Populus trichocarpa , a biofuel feedstock and model woody plant species. Despite their growing importance in plant genetics and metabolomics, few mGWASs are experimentally validated. Here, we present a functional genomics workflow for validating mGWAS-predicted enzyme–substrate relationships. We focus on uridine diphosphate–glycosyltransferases (UGTs), a large family of enzymes that catalyze sugar transfer to a variety of plant secondary metabolites involved in defense, signaling, and lignification. Glycosylation influences physiological roles, localization within cells and tissues, and metabolic fates of these metabolites. UGTs have substantially expanded in P. trichocarpa , presenting a challenge for large-scale characterization. Using a high-throughput assay, we produced substrate acceptance profiles for 40 previously uncharacterized candidate enzymes. Assays confirmed 10 of 13 leaf mGWAS associations, and a focused metabolite screen demonstrated varying levels of substrate specificity among UGTs. A substrate binding model case study of UGT-23 rationalized observed enzyme activities and mGWAS associations, including glycosylation of trichocarpinene to produce trichocarpin, a major higher-order salicylate in P. trichocarpa. We identified UGTs putatively involved in lignan, flavonoid, salicylate, and phytohormone metabolism, with potential implications for cell wall biosynthesis, nitrogen uptake, and biotic and abiotic stress response that determine sustainable biomass crop production. Our results provide new support for in silico analyses and evidence-based guidance for in vivo functional characterization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Assembly, comparative analysis, and utilization of a single haplotype reference genome for soybean

Cultivar Williams 82 has served as the reference genome for the soybean research community since 2008, but is known to have areas of genomic heterogeneity among different sub-lines. This work provides an updated assembly (version Wm82.a6) derived from a specific sub-line known as Wm82-ISU-01 (seeds available under USDA accession PI 704477). The genome was assembled using Pacific BioSciences HiFi reads and integrated into chromosomes using HiC. The 20 soybean chromosomes assembled into a genome of 1.01Gb, consisting of 36 contigs. The genome annotation identified 48 387 gene models, named in accordance with previous assembly versions Wm82.a2 and Wm82.a4. Comparisons of Wm82.a6 with other near-gapless assemblies of Williams 82 reveal large regions of genomic heterogeneity, including regions of differential introgression from the cultivar Kingwa within approximately 30 Mb and 25 Mb segments on chromosomes 03 and 07, respectively. Additionally, our analysis revealed a previously unknown large (> 20 Mb) heterogeneous region in the pericentromeric region of chromosome 12, where Wm82.a6 matches the ‘Williams’ haplotype while the other two near-gapless assemblies do not match the haplotype of either parent of Williams 82. In addition to the Wm82.a6 assembly, we also assembled the genome of ‘Fiskeby III,’ a rich resource for abiotic stress resistance genes. A genome comparison of Wm82.a6 with Fiskeby III revealed the nucleotide and structural polymorphisms between the two genomes within a QTL region for iron deficiency chlorosis resistance. The Wm82.a6 and Fiskeby III genomes described here will enhance comparative and functional genomics capacities and applications in the soybean community.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Populus PtrbHLH011 Is a Transcriptional Co‐Regulator Involved in the Activation of Cell Wall Biosynthesis by Iron Deprivation

The lack of a mechanistic understanding of the environmental plasticity of secondary cell wall (SCW) biosynthesis restricts large-scale biomass and bioenergy production on marginal lands. Using Populus (poplar), a key bioenergy crop, we discovered that iron deprivation, a prevalent abiotic stress on marginal lands, stimulates SCW biosynthesis in stems. We identified the transcription factor PtrbHLH011 as a critical regulator underlying this response. Through integrated analyses involving phenotypic characterisation of PtrbHLH011 knockout and overexpression plants, functional genomics and molecular investigations, we established that PtrbHLH011 functions as a central regulator of SCW biosynthesis, iron homeostasis and flavonoid biosynthesis by directly repressing essential genes in these pathways. Iron deprivation downregulates PtrbHLH011 expression, subsequently activating these biosynthetic pathways. Notably, cytosine base editing-based knockout of PtrbHLH011 significantly enhanced plant growth, yielding up to a 110% increase in stem diameter and a 300% increase in leaf iron content. These findings present a novel regulatory mechanism linking environmental iron availability to SCW biosynthesis and illustrate a practical strategy to improve biomass yield on iron-deficient marginal lands. Furthermore, our mechanistic insights into PtrbHLH011 target recognition and regulation provide a valuable foundation for precise manipulation of gene regulatory networks, facilitating the development of high-performance bioenergy crops adapted to marginal environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Inferring demographic and selective histories from population genomic data using a 2-step approach in species with coding-sparse genomes: an application to human data

Abstract The demographic history of a population, and the distribution of fitness effects (DFE) of newly arising mutations in functional genomic regions, are fundamental factors dictating both genetic variation and evolutionary trajectories. Although both demographic and DFE inference has been performed extensively in humans, these approaches have generally either been limited to simple demographic models involving a single population, or, where a complex population history has been inferred, without accounting for the potentially confounding effects of selection at linked sites. Taking advantage of the coding-sparse nature of the genome, we propose a 2-step approach in which coalescent simulations are first used to infer a complex multi-population demographic model, utilizing large non-functional regions that are likely free from the effects of background selection. We then use forward-in-time simulations to perform DFE inference in functional regions, conditional on the complex demography inferred and utilizing expected background selection effects in the estimation procedure. Throughout, recombination and mutation rate maps were used to account for the underlying empirical rate heterogeneity across the human genome. Importantly, within this framework it is possible to utilize and fit multiple aspects of the data, and this inference scheme represents a generalized approach for such large-scale inference in species with coding-sparse genomes.

Soni, Vivak (ORCID:0000000294969562)↗
DOE OSTI2024

High phenotypic and genotypic plasticity among strains of the mushroom-forming fungus Schizophyllum commune

Schizophyllum commune is a mushroom-forming fungus notable for its distinctive fruiting bodies with split gills. It is used as a model organism to study mushroom development, lignocellulose degradation and mating type loci. It is a hypervariable species with considerable genetic and phenotypic diversity between the strains. In this study, we systematically phenotyped 16 dikaryotic strains for aspects of mushroom development and 18 monokaryotic strains for lignocellulose degradation. There was considerable heterogeneity among the strains regarding these phenotypes. The majority of the strains developed mushrooms with varying morphologies, although some strains only grew vegetatively under the tested conditions. Growth on various carbon sources showed strain-specific profiles. The genomes of seven monokaryotic strains were sequenced and analyzed together with six previously published genome sequences. Moreover, the related species Schizophyllum fasciatum was sequenced. Although there was considerable genetic variation between the genome assemblies, the genes related to mushroom formation and lignocellulose degradation were well conserved. These sequenced genomes, in combination with the high phenotypic diversity, will provide a solid basis for functional genomics analyses of the strains of S. commune.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Lake Arrowhead Microbial Genomics Conference

The Lake Arrowhead Microbial Genomics Conference (formerly the E. coli and Small Genomes Conference, and the Microbial Genomes Conference) was held September 11-15, 2022, at the UCLA Conference Center at Lake Arrowhead, California. This conference is part of a yearly meeting initiated in 1991 to bring together genome sequencers, bioinformatics specialists, biologists, and geneticists, to forge interactions that would result in meaningful functional genomics. The goal of the meeting was to translate the influx of new genome sequencing information into useful biological studies. The Lake Arrowhead 2022 meeting had a major focus on microbial communities, the human microbiome, pathogens, phage therapy, bioinformatic methods of analysis, bioenergenics, and environmental microbiology. The field of genomics has reached the point where deriving the sequence of an organism’s entire genome is now seen as a beginning rather than an endpoint. Moreover, understanding the sequences of whole communities, and particularly those that make up different microbiomes, is now a central point of the field. The 2022 meeting covered microorganisms for which extensive analyses exist, and those for which new biological and technical strategies are being developed. The focus on biodiversity, the human microbiome, pathogenic organisms and methods of countering them, and bioenergetics added special significance to this meeting. This meeting was designed to have a mix of 36 invited presentations and poster sessions with more than 80 posters, and had 147 participants. This meeting has become established as a major annual microbial genomics conference during the last 29 years and has assured status and quality. It offers a major opportunity for young investigators and students to attend, present posters, and give talks. In fact, 50% of the invited speakers were early career stage scientists. An important impact of the conference was the forging of new collaborations between scientists and particularly young scientists with the array of multidisciplinary researchers at the meeting.

Miller, Jeffrey H.↗
DOE OSTI2022

Multiomic atlas with functional stratification and developmental dynamics of zebrafish cis -regulatory elements

Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center (https://danio-code.zfin.org) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

merlin , an improved framework for the reconstruction of high-quality genome-scale metabolic models

Abstract Genome-scale metabolic models have been recognised as useful tools for better understanding living organisms’ metabolism. merlin (https://www.merlin-sysbio.org/) is an open-source and user-friendly resource that hastens the models’ reconstruction process, conjugating manual and automatic procedures, while leveraging the user's expertise with a curation-oriented graphical interface. An updated and redesigned version of merlin is herein presented. Since 2015, several features have been implemented in merlin, along with deep changes in the software architecture, operational flow, and graphical interface. The current version (4.0) includes the implementation of novel algorithms and third-party tools for genome functional annotation, draft assembly, model refinement, and curation. Such updates increased the user base, resulting in multiple published works, including genome metabolic (re-)annotations and model reconstructions of multiple (lower and higher) eukaryotes and prokaryotes. merlin version 4.0 is the only tool able to perform template based and de novo draft reconstructions, while achieving competitive performance compared to state-of-the art tools both for well and less-studied organisms.

Capela, João (ORCID:0000000212352922)↗
DOE OSTI2025

Through the lens of bioenergy crops: advances, bottlenecks, and promises of plant engineering

Advances in engineering of bioenergy crops were driven over the past years by adapting technological breakthroughs and accelerating conventional applications but also exposed intriguing challenges. New tools revealed rich interconnectivity in the exponentially growing and dynamic 'big' omics data' of metabolomes, transcriptomes, and genomes at previously inaccessible magnitude (global, cross-species, meta-) and resolution (single cell). Insights enabled fresh hypotheses and stimulated disciplines such as functional genomics with discovery of broad regulatory networks and their determinants, that is, DNA parts, including promoters, regulatory elements, and transcription factors. Their rational design, assembly into increasingly complex blueprints, and installation into diverse chassis is an existing frontier that may benefit from emerging technologies to address bottlenecks. Interweaving nature-inspired to fully synthetic parts has already allowed building of fine-tuned regulatory circuits, or new-to-nature metabolic routes insulated from the biological context of the chassis species. Similarly, developments and the evolving need for unifying principles in plant transformation and species-agnostic technologies highlight future opportunities for engineering the next generation of bioenergy plants.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Supporting Information for manuscript: “A latitudinal gradient in S/G lignin monomer ratio driven by laccase in natural poplar variants”

Lignin composition plays a crucial role in plant structural integrity and environmental adaptation. However, the genetic and molecular mechanisms underlying natural variation in lignin composition remain poorly understood. This study investigates the syringyl-to-guaiacyl (S/G) lignin monomer ratio across a natural population of Populus trichocarpa spanning a latitudinal gradient along the Northwest coast of North America. By integrating biochemical, genomic, and geographic analysis, we identify key gene variants associated with S/G ratio differences. These datasets provide valuable insights into the evolutionary and functional genomics of lignin composition and serve as a resource for developing poplar variants optimized for forestry and bioenergy applications.

Poplar, lignin composition, laccases, latitude, ad↗
DOE OSTI2024

Phylometagenomics of cycad coralloid roots reveals shared symbiotic signals

Cycads are known to host symbiotic cyanobacteria, includingNostocalesspecies, as well as other sympatric bacterial taxa within their specialized coralloid roots. Yet, it is unknown if these bacteria share a phylogenetic origin and/or common genomic functions that allow them to engage in facultative symbiosis with cycad roots. To address this, we obtained metagenomic sequences from 39 coralloid roots sampled from diverse cycad species and origins in Australia and Mexico. Culture-independent shotgun metagenomic sequencing was used to validate sub-community co-cultures as an efficient approach for functional and taxonomic analysis. Our metanalysis shows a host-independent microbiome core consisting of seven bacterial orders with high species diversity within the identified taxa. Moreover, we recovered 43 cyanobacterial metagenome-assembled genomes, and in addition toNostocspp., symbiotic cyanobacteria of the genusAulosirawere identified for the first time. Using this robust dataset, we used phylometagenomic analysis to reveal three monophyletic cyanobiont clades, two host-generalist and one cycad-specific that includesAulosiraspp. Although the symbiotic clades have independently arisen, they are enriched in certain functional genes, such as those related to secondary metabolism. Furthermore, the taxonomic composition of associated sympatric bacterial taxa remained constant. Our research quadruples the number of cycad cyanobiont genomes and provides a robust framework to decipher cyanobacterial symbioses, with the potential of improving our understanding of symbiotic communities. This study lays a solid foundation to harness cyanobionts for agriculture and bioprospection, and assist in conservation of critically endangered cycads.

Genetics & Heredity↗
DOE OSTI2023

Haplotype‐resolved genome assembly of Populus tremula × P. alba reveals aspen‐specific megabase satellite DNA

SUMMARY Populus species play a foundational role in diverse ecosystems and are important renewable feedstocks for bioenergy and bioproducts. Hybrid aspen Populus tremula × P. alba INRA 717‐1B4 is a widely used transformation model in tree functional genomics and biotechnology research. As an outcrossing interspecific hybrid, its genome is riddled with sequence polymorphisms which present a challenge for sequence‐sensitive analyses. Here we report a telomere‐to‐telomere genome for this hybrid aspen with two chromosome‐scale, haplotype‐resolved assemblies. We performed a comprehensive analysis of the repetitive landscape and identified both tandem repeat array‐based and array‐less centromeres. Unexpectedly, the most abundant satellite repeats in both haplotypes lie outside of the centromeres, consist of a 147 bp monomer PtaM147, frequently span >1 megabases, and form heterochromatic knobs. PtaM147 repeats are detected exclusively in aspens (section Populus ) but PtaM147‐like sequences occur in LTR‐retrotransposons of closely related species, suggesting their origin from the retrotransposons. The genomic resource generated for this transformation model genotype has greatly improved the design and analysis of genome editing experiments that are highly sensitive to sequence polymorphisms. The work should motivate future hypothesis‐driven research to probe into the function of the abundant and aspen‐specific PtaM147 satellite DNA.

Zhou, Ran↗
DOE OSTI2023

Targeted mutagenesis with sequence–specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

Many of the world's most important crops are polyploid. The presence of more than two sets of chromosomes within their nuclei and frequently aberrant reproductive biology in polyploids present obstacles to conventional breeding. The presence of a larger number of homoeologous copies of each gene makes random mutation breeding a daunting task for polyploids. Genome editing has revolutionized improvement of polyploid crops as multiple gene copies and/or alleles can be edited simultaneously while preserving the key attributes of elite cultivars. Most genome–editing platforms employ sequence–specific nucleases (SSNs) to generate DNA double–stranded breaks at their target gene. Such DNA breaks are typically repaired via the error–prone nonhomologous end–joining process, which often leads to frame shift mutations, causing loss of gene function. Genome editing has enhanced the disease resistance, yield components, and end–use quality of polyploid crops. However, identification of candidate targets, genotyping, and requirement of high mutagenesis efficiency remain bottlenecks for targeted mutagenesis in polyploids. In this review, we will survey the tremendous progress of SSN–mediated targeted mutagenesis in polyploid crop improvement, discuss its challenges, and identify optimizations needed to sustain further progress.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

BSMV-mediated genome editing exhibits host-specific heritability: germline transmission in barley and somatic edits in Nicotiana benthamiana

Plant RNA virus–mediated guide RNA (gRNA) delivery represents a transformative advance in genome editing technologies. Unlike conventional transformation methods that rely on labor-intensive tissue culture and regeneration for each individual gRNA delivery, viral vectors can rapidly and systemically transmit gRNAs into pre-established Cas-expressing plants, providing an accelerated route for functional genomics and trait discovery directly in planta . However, key design parameters, including subgenomic promoter choice, transcript architecture, and their effects on viral fitness and editing outcomes, remain to be elucidated for most viral platforms. We developed five Barley stripe mosaic virus (BSMV) vectors, each with distinct subgenomic promoter elements to drive single gRNA expression. These were initially evaluated in Cas9-expressing transgenic Nicotiana benthamiana plants targeting the Phytoene desaturase ( PDS ) gene to compare their editing efficiencies. Single gRNAs expressed under the duplicated γb subgenomic promoter or when fused directly to the γb genome achieved the highest mutation frequencies (up to 90% at 60 days post-inoculation), whereas β1- and β2-driven sgRNAs produced delayed and reduced editing. Thus, promoter selection critically determines gRNA accumulation and the efficacy of BSMV-mediated genome editing. The top-performing design was then applied to Cas9-expressing barley ( Hordeum vulgare ) targeting HvCMF7 (conferring green-white variegation) and HvGW2.1 (impacts grain width and weight). BSMV spread systemically throughout barley, inducing somatic and heritable mutations at frequencies up to 100%, with virus-free edited progeny. In contrast, despite robust somatic editing in N. benthamiana, no heritable mutations were detected indicating species-dependent limitations in germline transmission. Our systematic comparison of subgenomic promoter architectures establishes clear design principles for optimizing viral vector–mediated delivery. Promoter choice and transcript structure critically shape editing efficiency and viral stability. The host-specific boundary for germline editing, defined by efficient heritable editing in barley but not N. benthamiana , highlights where BSMV offers advantages and where alternative vectors or hybrid strategies are required, guiding rational platform selection for diverse crop species and applications. Collectively, these findings establish BSMV as a promising next-generation vector for rapid, tissue culture–free, and transformation-independent genome editing in cereals and other recalcitrant monocots.

barley↗
DOE OSTI2024

Soybean genomics research community strategic plan: A vision for 2024–2028

Abstract This strategic plan summarizes the major accomplishments achieved in the last quinquennial by the soybean [Glycine max(L.) Merr.] genetics and genomics research community and outlines key priorities for the next 5 years (2024–2028). This work is the result of deliberations among over 50 soybean researchers during a 2‐day workshop in St Louis, MO, USA, at the end of 2022. The plan is divided into seven traditional areas/disciplines: Breeding, Biotic Interactions, Physiology and Abiotic Stress, Functional Genomics, Biotechnology, Genomic Resources and Datasets, and Computational Resources. One additional section was added, Training the Next Generation of Soybean Researchers, when it was identified as a pressing issue during the workshop. This installment of the soybean genomics strategic plan provides a snapshot of recent progress while looking at future goals that will improve resources and enable innovation among the community of basic and applied soybean researchers. We hope that this work will inform our community and increase support for soybean research.

Genetics & Heredity↗
DOE OSTI2024

Diverse signatures of convergent evolution in cactus-associated yeasts

Many distantly related organisms have convergently evolved traits and lifestyles that enable them to live in similar ecological environments. However, the extent of phenotypic convergence evolving through the same or distinct genetic trajectories remains an open question. Here, we leverage a comprehensive dataset of genomic and phenotypic data from 1,049 yeast species in the subphylum Saccharomycotina (Kingdom Fungi, Phylum Ascomycota) to explore signatures of convergent evolution in cactophilic yeasts, ecological specialists associated with cacti. We inferred that the ecological association of yeasts with cacti arose independently approximately 17 times. Using a machine learning–based approach, we further found that cactophily can be predicted with 76% accuracy from both functional genomic and phenotypic data. The most informative feature for predicting cactophily was thermotolerance, which we found to be likely associated with altered evolutionary rates of genes impacting the cell envelope in several cactophilic lineages. We also identified horizontal gene transfer and duplication events of plant cell wall–degrading enzymes in distantly related cactophilic clades, suggesting that putatively adaptive traits evolved independently through disparate molecular mechanisms. Notably, we found that multiple cactophilic species and their close relatives have been reported as emerging human opportunistic pathogens, suggesting that the cactophilic lifestyle—and perhaps more generally lifestyles favoring thermotolerance—might preadapt yeasts to cause human disease. This work underscores the potential of a multifaceted approach involving high-throughput genomic and phenotypic data to shed light onto ecological adaptation and highlights how convergent evolution to wild environments could facilitate the transition to human pathogenicity.

59 BASIC BIOLOGICAL SCIENCES↗