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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Mycorrhiza‐induced mycocypins of Laccaria bicolor are potent protease inhibitors with nematotoxic and collembola antifeedant activity

Fungivory of mycorrhizal hyphae has a significant impact on fungal fitness and, by extension, on nutrient transfer between fungi and host plants in natural ecosystems. Mycorrhizal fungi have therefore evolved an arsenal of chemical compounds that are hypothesized to protect the hyphal tissues from being eaten, such as the protease inhibitors mycocypins. The genome of the ectomycorrhizal fungus Laccaria bicolor has an unusually high number of mycocypin-encoding genes. We have characterized the evolution of this class of proteins, identified those induced by symbiosis with a host plant and characterized the biochemical properties of two upregulated L. bicolor mycocypins. More than half of L. bicolor mycocypin-encoding genes are differentially expressed during symbiosis or fruiting body formation. We show that two L. bicolor mycocypins that are strongly induced during symbiosis are cysteine protease inhibitors and exhibit similar but distinct localization in fungal tissues at different developmental stages and during interaction with a host plant. Moreover, we show that these L. bicolor mycocypins have toxic and feeding deterrent effect on nematodes and collembolans, respectively. Therefore, L. bicolor mycocypins may be part of a mechanism by which this species deters grazing by different members of the soil food web.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Desert truffle genomes reveal their reproductive modes and new insights into plant–fungal interaction and ectendomycorrhizal lifestyle

Desert truffles are edible hypogeous fungi forming ectendomycorrhizal symbiosis with plants of Cistaceae family. Knowledge about the reproductive modes of these fungi and the molecular mechanisms driving the ectendomycorrhizal interaction is lacking. Genomes of the highly appreciated edible desert truffles Terfezia claveryi Chatin and Tirmania nivea Trappe have been sequenced and compared with other Pezizomycetes. Transcriptomes of T. claveryi × Helianthemum almeriense mycorrhiza from well-watered and drought-stressed plants, when intracellular colonizations is promoted, were investigated. We have identified the fungal genes related to sexual reproduction in desert truffles and desert-truffles-specific genomic and secretomic features with respect to other Pezizomycetes, such as the expansion of a large set of gene families with unknown Pfam domains and a number of species or desert-truffle-specific small secreted proteins differentially regulated in symbiosis. A core set of plant genes, including carbohydrate, lipid-metabolism, and defence-related genes, differentially expressed in mycorrhiza under both conditions was found. Our results highlight the singularities of desert truffles with respect to other mycorrhizal fungi while providing a first glimpse on plant and fungal determinants involved in ecto to endo symbiotic switch that occurs in desert truffle under dry conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Transcriptional, metabolic, physiological and developmental responses of switchgrass to phosphorus limitation

Abstract Knowing how switchgrass ( Panicum virgatum L . ) responds and adapts to phosphorus (P)‐limitation will aid efforts to optimize P acquisition and use in this species for sustainable biomass production. This integrative study investigated the impacts of mild, moderate, and severe P‐stress on genome transcription and whole‐plant metabolism, physiology and development in switchgrass. P‐limitation reduced overall plant growth, increased root/shoot ratio, increased root branching at moderate P‐stress, and decreased root diameter with increased density and length of root hairs at severe P‐stress. RNA‐seq analysis revealed thousands of genes that were differentially expressed under moderate and severe P‐stress in roots and/or shoots compared to P‐replete plants, with many stress‐induced genes involved in transcriptional and other forms of regulation, primary and secondary metabolism, transport, and other processes involved in P‐acquisition and homeostasis. Amongst the latter were multiple miRNA399 genes and putative targets of these. Metabolite profiling showed that levels of most sugars and sugar alcohols decreased with increasing P stress, while organic and amino acids increased under mild and moderate P‐stress in shoots and roots, although this trend reversed under severe P‐stress, especially in shoots.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

De novo transcriptome sequencing of Capsicum frutescens . L and comprehensive analysis of salt stress alleviating mechanism by Bacillus atrophaeus WU ‐9

Abstract Salt stress, as one of the most severe environmental stresses, can cause a series of changes in plants. However, the explanation of plant salt stress alleviating mechanism of plant growth–promoting rhizobacteria (PGPR) was hindered by the limited availability of transcriptomic information for salt stress‐treated plants grown in a microorganism‐controlled environment. Our previous reports have selected Bacillus atrophaeus WU‐9 as PGPR significantly alleviating pepper ( Capsicum frutescens . L) salt stress. In this work, the RNA‐seq analysis of salt stress‐treated and untreated plants, grown with and without WU‐9 in a microorganism‐controlled environment, was used to reveal the plant salt stress alleviating mechanisms of WU‐9. Twelve sequencing libraries, prepared by treating with WU‐9 and salt (150 mM NaCl for 36 h), were constructed by RNA‐Seq technique. Non‐inoculated seedlings mainly respond to salt stress through regulation of signal transduction, such as ethylene‐activated signaling pathway, signaling and cell communication, etc. And ethylene signal participated in salt stress response in pepper through regulating defense responses, fruit ripening and senescence. WU‐9 inoculation under salt stress mainly improves salt tolerance and plant growth by regulating salt stress‐responding ethylene and auxin signal transduction, utilization of proline, photosynthesis, antioxidant enzyme activities and cell enlargement. Furthermore, 86 differentially expressed genes and 20 transcription factors were identified as associated with salt stress response and tolerance. Thus, this innovative transcriptomic study identified the salt stress response and alleviation in C. frutescens . L with PGPR inoculation. This result provided novel insights into the salinity alleviation in pepper regulated by PGPR.

Wang, Wenfei↗
DOE OSTI2025

Hidden diversity: Transcriptomic and photosynthetic variation among common ‘wild type’ Chlamydomonas strains

The unicellular green alga Chlamydomonas reinhardtii is a widely studied reference organism, particularly in photosynthesis research. It employs photoprotective mechanisms, such as state transitions (ST) and non-photochemical quenching (NPQ), to cope with rapid light changes. Most widely used strains share a recent common ancestor yet differ by up to ~50 000 nuclear variants—genetic diversity that is often overlooked. Even among ‘wild type’ strains, we document significant phenotypic differences, such as pigment accumulation, and nutrient utilization. To elucidate the basis for this variation, we compared transcriptomes and physiological traits of seven commonly used laboratory strains, including the reference strain and the CLiP mutant library parental strain. Despite identical growth conditions, ~40% of genes were differentially expressed between strains. Most of these differences are attributable to changes that have accrued during laboratory propagation, and adverse conditions may have driven transcriptomic drift. At the physiological level, we catalog the range of strain-dependent responses related to photosynthesis and high light (HL) acclimation. Specifically, (i) all strains develop NPQ upon HL exposure, but to various degrees, (ii) they show a substantial variation in ST capacity, and (iii) they regulate the composition of the photosynthetic apparatus differently. We find that NPQ levels do not correlate with LHCSR3 expression, suggesting an additional layer of NPQ regulation. STs are constantly activated and independent of growth light intensities. Overall, our findings highlight significant strain-to-strain differences in virtually all photosynthetic parameters, emphasizing the importance of careful strain selection in future research endeavors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Compilation and utilization of a sorghum transcriptome compendium for gene regulatory network analysis and crop trait engineering

Sorghum bicolor (Sorghum) is a drought and heat tolerant C4 grass crop used to produce grain, forage, biofuels, and other bioproducts. Genetic improvement of sorghum hybrid crops is aided by a large and diverse germplasm, sorghum's diploid inbreeding genetics, and a relatively small genome that has facilitated genomic research. Over the past 20 years, the sorghum research community characterized the cytogenetic and recombinant landscapes of sorghum's 10 chromosomes, sequenced and annotated the sorghum genome, and used that information to identify genes/alleles that modulate flowering time, plant height, seed shattering, and other important traits. More recently, >1000 RNA-seq transcriptome profiles were collected from 15 sorghum genotypes to help understand the genetic basis of variation in growth and development of sorghum stems, tillers, roots, and leaves, and the regulation of biosynthetic pathways that produce epicuticular wax, dhurrin, and RFOs, compounds that contribute to sorghum's resilience. Transcriptome studies were designed to identify differentially expressed genes that are co-expressed during development or in response to a treatment to enable construction of gene regulatory networks. Co-expression and network analysis identified transcription factors and their cognate binding sites in target gene promoters and signaling pathways that modulate gene regulatory networks providing gene editing targets for further trait optimization. RNA-seq data from >20 experiments targeting sorghum organs, tissues, cell types, developmental stages, and responses to environmental conditions (i.e., diel, day-length, shading, water-deficit, temperature) has been compiled in a sorghum transcriptome compendium. The goal of this resource paper is to describe compendium content, accessibility, and a compendium data analysis pipeline and to illustrate the types of information that can be derived from the compendium with a focus on the elucidation of gene regulatory networks useful for guiding the improvement of sorghum traits through gene editing.

RNA-seq↗
DOE OSTI2024

An alternate route for cellulose microfibril biosynthesis in plants

Similar to cellulose synthases (CESAs), cellulose synthase–like D (CSLD) proteins synthesize β-1,4-glucan in plants. CSLDs are important for tip growth and cytokinesis, but it was unknown whether they form membrane complexes in vivo or produce microfibrillar cellulose. We produced viable CESA-deficient mutants of the moss Physcomitrium patens to investigate CSLD function without interfering CESA activity. Microscopy and spectroscopy showed that CESA-deficient mutants synthesize cellulose microfibrils that are indistinguishable from those in vascular plants. Correspondingly, freeze-fracture electron microscopy revealed rosette-shaped particle assemblies in the plasma membrane that are indistinguishable from CESA-containing rosette cellulose synthesis complexes (CSCs). Our data show that proteins other than CESAs, most likely CSLDs, produce cellulose microfibrils in P. patens protonemal filaments. The data suggest that the specialized roles of CSLDs in cytokinesis and tip growth are based on differential expression and different interactions with microtubules and possibly Ca 2+ , rather than structural differences in the microfibrils they produce.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Capturing an Early Gene Induction Event during Wood Decay by the Brown Rot Fungus Rhodonia placenta

Brown rot fungi dominate wood decomposition in coniferous forests, and their carbohydrate-selective mechanisms are of commercial interest. Brown rot was recently described as a two-step, sequential mechanism orchestrated by fungi using differentially expressed genes (DEGs) and consisting of oxidation via reactive oxygen species (ROS) followed by enzymatic saccharification. There have been indications, however, that the initial oxidation step itself might require induction. To capture this early gene regulation event, here, we integrated fine-scale cryosectioning with whole-transcriptome sequencing to dissect gene expression at the single-hyphal-cell scale (tens of micrometers). This improved the spatial resolution 50-fold, relative to previous work, and we were able to capture the activity of the first 100 μm of hyphal front growth by Rhodonia placenta in aspen wood. This early decay period was dominated by delayed gene expression patterns as the fungus ramped up its mechanism. These delayed DEGs included many genes implicated in ROS pathways (lignocellulose oxidation [LOX]) that were previously and incorrectly assumed to be constitutively expressed. These delayed DEGs, which include those with and without predicted functions, also create a focused subset of target genes for functional genomics. However, this delayed pattern was not universal, with a few genes being upregulated immediately at the hyphal front. Most notably, this included a gene commonly implicated in hydroquinone and iron redox cycling: benzoquinone reductase.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Evidence for autotrophic growth of purple sulfur bacteria using pyrite as electron and sulfur source

ABSTRACT Purple sulfur bacteria (PSB) are capable of anoxygenic photosynthesis via oxidizing reduced sulfur compounds and are considered key drivers of the sulfur cycle in a range of anoxic environments. In this study, we show that Allochromatium vinosum (a PSB species) is capable of autotrophic growth using pyrite as the electron and sulfur source. Comparative growth profile, substrate characterization, and transcriptomic sequencing data provided valuable insight into the molecular mechanisms underlying the bacterial utilization of pyrite and autotrophic growth. Specifically, the pyrite-supported cell cultures (“py”’) demonstrated robust but much slower growth rates and distinct patterns from their sodium sulfide-amended positive controls. Up to ~200-fold upregulation of genes encoding various c - and b -type cytochromes was observed in “py,” pointing to the high relevance of these molecules in scavenging and relaying electrons from pyrite to cytoplasmic metabolisms. Conversely, extensive downregulation of genes related to LH and RC complex components indicates that the electron source may have direct control over the bacterial cells’ photosynthetic activity. In terms of sulfur metabolism, genes encoding periplasmic or membrane-bound proteins (e.g., FccAB and SoxYZ) were largely upregulated, whereas those encoding cytoplasmic proteins (e.g., Dsr and Apr groups) are extensively suppressed. Other notable differentially expressed genes are related to flagella/fimbriae/pilin(+), metal efflux(+), ferrienterochelin(−), and [NiFe] hydrogenases(+). Characterization of the biologically reacted pyrite indicates the presence of polymeric sulfur. These results have, for the first time, put the interplay of PSB and transition metal sulfide chemistry under the spotlight, with the potential to advance multiple fields, including metal and sulfur biogeochemistry, bacterial extracellular electron transfer, and artificial photosynthesis. IMPORTANCE Microbial utilization of solid-phase substrates constitutes a critical area of focus in environmental microbiology, offering valuable insights into microbial metabolic processes and adaptability. Recent advancements in this field have profoundly deepened our knowledge of microbial physiology pertinent to these scenarios and spurred innovations in biosynthesis and energy production. Furthermore, research into interactions between microbes and solid-phase substrates has directly linked microbial activities to the surrounding mineralogical environments, thereby enhancing our understanding of the relevant biogeochemical cycles. Our study represents a significant step forward in this field by demonstrating, for the first time, the autotrophic growth of purple sulfur bacteria using insoluble pyrite (FeS2) as both the electron and sulfur source. The presented comparative growth profiles, substrate characterizations, and transcriptomic sequencing data shed light on the relationships between electron donor types, photosynthetic reaction center activities, and potential extracellular electron transfer in these organisms capable of anoxygenic photosynthesis. Furthermore, the findings of our study may provide new insights into early-Earth biogeochemical evolutions, offering valuable constraints for understanding the environmental conditions and microbial processes that shaped our planet’s history.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Alternate routes to acetate tolerance lead to varied isoprenol production from mixed carbon sources in Pseudomonas putida

ABSTRACT Lignocellulose is a renewable resource for the production of a diverse array of platform chemicals, including the biofuel isoprenol. Although this carbon stream provides a rich source of sugars, other organic compounds, such as acetate, can be used by microbial hosts. Here, we examined the growth and isoprenol production in a Pseudomonas putida strain pre-tolerized (“PT”) background where its native isoprenol catabolism pathway is deleted, using glucose and acetate as carbon sources. We found that PT displays impaired growth in minimal medium containing acetate and often fails to grow in glucose-acetate medium. Using a mutant recovery-based approach, we generated tolerized strains that overcame these limitations, achieving fast growth and isoprenol production in the mixed carbon feed. Changes in the glucose and acetate assimilation routes, including an upregulation in PP_0154 (SpcC, succinyl-CoA:acetate CoA-transferase) and differential expression of the gluconate assimilation pathways, were key for higher isoprenol titers in the tolerized strains, whereas a different set of mechanisms were likely enabling tolerance phenotypes in media containing acetate. Among these, a coproporphyrinogen-III oxidase (HemN) was upregulated across all tolerized strains and in one isolate required for acetate tolerance. Utilizing a defined glucose and acetate mixture ratio reflective of lignocellulosic feedstocks for isoprenol production in P. putida allowed us to obtain insights into the dynamics and challenges unique to dual carbon source utilization that are obscured when studied separately. Together, this enabled the development of a P. putida bioconversion chassis able to use a more complex carbon stream to produce isoprenol. IMPORTANCE Acetate is a relatively abundant component of many lignocellulosic carbon streams and has the potential to be used together with sugars, especially in microbes with versatile catabolism such as P. putida . However, the use of mixed carbon streams necessitates additional optimization. Furthermore, the use of P. putida for the production of the biofuel target, isoprenol, requires the use of engineered strains that have additional growth and production constraints when cultivated in acetate and glucose mixtures. In this study, we generate acetate-tolerant P. putida strains that overcome these challenges and examine their ability to produce isoprenol. We show that acetate tolerance and isoprenol production, although independent phenotypes, can both be optimized in a given P. putida strain. Using proteomics and whole genome sequencing, we examine the molecular basis of both phenotypes and show that tolerance to acetate can occur via alternate routes and result in different impacts on isoprenol production.

de Siqueira, Guilherme M. V. (ORCID:00000002364563↗
DOE OSTI2026

Transcriptomic and functional analyses uncover a conserved effector driving genotype-dependent virulence in the Sphaerulina musiva-Populus trichocarpa interaction

The introduction of invasive microbes compromises the structure, biodiversity, and function of naïve ecosystems. Sphaerulina musiva, a hemibiotrophic pathogen that causes leaf spot and stem cankers in Populus species, exemplifies an invasive fungal pathogen spread by human activities. However, the genetic mechanisms of pathogenicity and virulence are poorly understood, impeding mitigation strategies. We utilized RNA sequencing to identify fungal effectors linked to stem canker formation, informing the development of future strategies for effective disease management. Our analysis revealed 70 genes differentially expressed at 2 weeks and 110 genes at 3 weeks between inoculated trees and controls. Notably, the gene with the highest expression at 2 weeks and the second highest at 3 weeks was homologous to Extracellular protein 2 (Ecp2). Complementary genome-wide association studies linked sequence polymorphisms in this locus to phenotypic variation in disease severity. Infiltration of S. musiva Ecp2 into Populus trichocarpa leaves induced necrosis in susceptible genotypes. Gene disruption using a CRISPR-Cas9 RNP system resulted in a genotype-dependent reduction of stem canker and disease severity. Tracing the evolutionary history of this effector across the fungal kingdom, we uncovered clade-specific gene-family expansions and orthologs in new species. These findings raise questions about the function and adaptive significance of these gene families in fungal lifestyles. Our study provides the first tractable target for breeding resistant poplar genotypes, addressing the challenges of managing S. musiva and uncovering mechanisms that drive its virulence, and provides deeper insights into the evolutionary dynamics of a conserved small-secreted protein with a diversity of functions.

Sondreli, Kelsey L [Oregon State University]↗
DOE OSTI2023

A Genome-Scale Atlas Reveals Complex Interplay of Transcription and Translation in an Archaeon

The scale of post-transcriptional regulation and the implications of its interplay with other forms of regulation in environmental acclimation are underexplored for organisms of the domain Archaea. Here, we have investigated the scale of post-transcriptional regulation in the extremely halophilic archaeon Halobacterium salinarum NRC-1 by integrating the transcriptome-wide locations of transcript processing sites (TPSs) and SmAP1 binding, the genome-wide locations of antisense RNAs (asRNAs), and the consequences of RNase_2099C knockout on the differential expression of all genes. This integrated analysis has discovered that 54% of all protein-coding genes in the genome of this haloarchaeon are likely targeted by multiple mechanisms for putative post-transcriptional processing and regulation, with about 20% of genes likely being regulated by combinatorial schemes involving SmAP1, asRNAs, and RNase_2099C. Comparative analysis of mRNA levels (transcriptome sequencing [RNA-Seq]) and protein levels (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry [SWATH-MS]) for 2,579 genes over four phases of batch culture growth in complex medium generated additional evidence for the conditional post-transcriptional regulation of 7% of all protein-coding genes. We demonstrate that post-transcriptional regulation may act to fine-tune specialized and rapid acclimation to stressful environments, e.g., as a switch to turn on gas vesicle biogenesis to promote vertical relocation under anoxic conditions and modulate the frequency of transposition by insertion sequence (IS) elements of the IS200/IS605, IS4, and ISH3 families. Findings from this study are provided as an atlas in a public Web resource (https://halodata.systemsbiology.net).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Novel Anti-LY6G6D/CD3 T-Cell–Dependent Bispecific Antibody for the Treatment of Colorectal Cancer

Abstract New therapeutics and combination regimens have led to marked clinical improvements for the treatment of a subset of colorectal cancer. Immune checkpoint inhibitors have shown clinical efficacy in patients with mismatch-repair–deficient or microsatellite instability–high (MSI-H) metastatic colorectal cancer (mCRC). However, patients with microsatellite-stable (MSS) or low levels of microsatellite instable (MSI-L) colorectal cancer have not benefited from these immune modulators, and the survival outcome remains poor for the majority of patients diagnosed with mCRC. In this article, we describe the discovery of a novel T-cell–dependent bispecific antibody (TDB) targeting tumor-associated antigen LY6G6D, LY6G6D-TDB, for the treatment of colorectal cancer. RNAseq analysis showed that LY6G6D was differentially expressed in colorectal cancer with high prevalence in MSS and MSI-L subsets, whereas LY6G6D expression in normal tissues was limited. IHC confirmed the elevated expression of LY6G6D in primary and metastatic colorectal tumors, whereas minimal or no expression was observed in most normal tissue samples. The optimized LY6G6D-TDB, which targets a membrane-proximal epitope of LY6G6D and binds to CD3 with high affinity, exhibits potent antitumor activity both in vitro and in vivo. In vitro functional assays show that LY6G6D-TDB–mediated T-cell activation and cytotoxicity are conditional and target dependent. In mouse xenograft tumor models, LY6G6D-TDB demonstrates antitumor efficacy as a single agent against established colorectal tumors, and enhanced efficacy can be achieved when LY6G6D-TDB is combined with PD-1 blockade. Our studies provide evidence for the therapeutic potential of LY6G6D-TDB as an effective treatment option for patients with colorectal cancer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Cell Population–resolved Multiomics Atlas of the Developing Lung

The lung is a vital organ that undergoes extensive morphological and functional changes during postnatal development. To disambiguate how different cell populations contribute to organ development, we performed proteomic and transcriptomic analyses of four sorted cell populations from the lung of human subjects aged 0 to 8 years-old with a focus on early life. The cell populations analyzed included epithelial, endothelial, mesenchymal, and immune cells. Our results revealed distinct molecular signatures for each of the sorted cell populations that enable the description of molecular shifts occurring in these populations during post-natal development. Here, we confirmed that the proteome of the different cell populations was distinct regardless of age and identified functions specific to each population. We identified a series of cell population protein markers, including those located at the cell surface, that show differential expression and distribution on RNA in situ hybridization and immunofluorescence imaging. We validated the spatial distribution of AT1 and endothelial cell surface markers. Temporal analyses of the proteome of each of the four populations revealed processes modulated during postnatal development and disambiguating results obtained on whole tissue proteome. Finally, the proteome was compared to a transcriptomics survey performed on the same lung samples to evaluate processes under post-transcriptional control.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Asc-Seurat: analytical single-cell Seurat-based web application

Abstract Background Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of transcriptomes, arising as a powerful tool for discovering and characterizing cell types and their developmental trajectories. However, scRNA-seq analysis is complex, requiring a continuous, iterative process to refine the data and uncover relevant biological information. A diversity of tools has been developed to address the multiple aspects of scRNA-seq data analysis. However, an easy-to-use web application capable of conducting all critical steps of scRNA-seq data analysis is still lacking. Summary We present Asc-Seurat, a feature-rich workbench, providing an user-friendly and easy-to-install web application encapsulating tools for an all-encompassing and fluid scRNA-seq data analysis. Asc-Seurat implements functions from the Seurat package for quality control, clustering, and genes differential expression. In addition, Asc-Seurat provides a pseudotime module containing dozens of models for the trajectory inference and a functional annotation module that allows recovering gene annotation and detecting gene ontology enriched terms. We showcase Asc-Seurat’s capabilities by analyzing a peripheral blood mononuclear cell dataset. Conclusions Asc-Seurat is a comprehensive workbench providing an accessible graphical interface for scRNA-seq analysis by biologists. Asc-Seurat significantly reduces the time and effort required to analyze and interpret the information in scRNA-seq datasets.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Horizontal gene transfer of the Mer operon is associated with large effects on the transcriptome and increased tolerance to mercury in nitrogen-fixing bacteria

Abstract Background Mercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Horizontal gene transfer has potential to confer immediate de novo adaptations to stress. We used comparative genomics of high quality de novo assemblies to identify structural differences in the genomes of nitrogen-fixing rhizobia that were isolated from a mercury (Hg) mine site that show high variation in their tolerance to Hg. Results Our analyses identified multiple structurally conserved merA homologs in the genomes of Sinorhizobium medicae and Rhizobium leguminosarum but only the strains that possessed a Mer operon exhibited 10-fold increased tolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while in bacteroids showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant. Transfer of a plasmid containing the Mer operon from the most tolerant strain to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the Mer operon is able to confer hyper tolerance to Hg. Conclusions Mer operons have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

BdERECTA controls vasculature patterning and phloem-xylem organization in Brachypodium distachyon

Background: The vascular system of plants consists of two main tissue types, xylem and phloem. These tissues are organized into vascular bundles that are arranged into a complex network running through the plant that is essential for the viability of land plants. Despite their obvious importance, the genes involved in the organization of vascular tissues remain poorly understood in grasses. Results: We studied in detail the vascular network in stems from the model grass Brachypodium distachyon (Brachypodium) and identified a large set of genes differentially expressed in vascular bundles versus parenchyma tissues. To decipher the underlying molecular mechanisms of vascularization in grasses, we conducted a forward genetic screen for abnormal vasculature. We identified a mutation that severely affected the organization of vascular tissues. This mutant displayed defects in anastomosis of the vascular network and uncommon amphivasal vascular bundles. The causal mutation is a premature stop codon in ERECTA, a LRR receptor-like serine/threonine-protein kinase. Mutations in this gene are pleiotropic indicating that it serves multiple roles during plant development. This mutant also displayed changes in cell wall composition, gene expression and hormone homeostasis. Conclusion: In summary, ERECTA has a pleiotropic role in Brachypodium. We propose a major role of ERECTA in vasculature anastomosis and vascular tissue organization in Brachypodium.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Cyanobacterial circadian regulation enhances bioproduction under subjective nighttime through rewiring of carbon partitioning dynamics, redox balance orchestration, and cell cycle modulation

Abstract Background The industrial feasibility of photosynthetic bioproduction using cyanobacterial platforms remains challenging due to insufficient yields, particularly due to competition between product formation and cellular carbon demands across different temporal phases of growth. This study investigates how circadian clock regulation impacts carbon partitioning between storage, growth, and product synthesis in Synechococcus elongatus PCC 7942, and provides insights that suggest potential strategies for enhanced bioproduction. Results After entrainment to light-dark cycles, PCC 7942 cultures transitioned to constant light revealed distinct temporal patterns in sucrose production, exhibiting three-fold higher productivity during subjective night compared to subjective day despite moderate down-regulation of genes from the photosynthetic apparatus. This enhanced productivity coincided with reduced glycogen accumulation and halted cell division at subjective night time, suggesting temporal separation of competing processes. Transcriptome analysis revealed coordinated circadian clock-driven adjustment of the cell cycle and rewiring of energy and carbon metabolism, with over 300 genes showing differential expression across four time points. The subjective night was characterized by altered expression of cell division-related genes and reduced expression of genes involved in glycogen synthesis, while showing upregulation of glycogen degradation pathways, alternative electron flow components, the pentose phosphate pathway, and oxidative decarboxylation of pyruvate. These molecular changes created favorable conditions for product formation through enhanced availability of major sucrose precursors (glucose-1-phosphate and fructose-6-phosphate) and maintained redox balance through multiple mechanisms. Conclusions Our analysis of circadian regulatory rewiring of carbon metabolism and redox balancing suggests two potential approaches that could be developed for improving cyanobacterial bioproduction: leveraging natural circadian rhythms for optimizing cultivation conditions and timing of pathway induction, and engineering strains that mimic circadian-driven metabolic shifts through controlled carbon flux redistribution and redox rebalancing. While these strategies remain to be tested, they could theoretically improve the efficiency of photosynthetic bioproduction by enabling better temporal separation between cell growth, carbon storage accumulation, and product synthesis phases.

59 BASIC BIOLOGICAL SCIENCES↗