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DOE OSTI2021

Examining the Relationship Between the Testate Amoeba Hyalosphenia papilio (Arcellinida, Amoebozoa) and its Associated Intracellular Microalgae Using Molecular and Microscopic Methods

Symbiotic relationships between heterotrophic and phototrophic partners are common in microbial eukaryotes. Among Arcellinida (Amoebozoa) several species are associated with microalgae of the genus Chlorella (Archaeplastida). So far, these symbioses were assumed to be stable and mutualistic, yet details of the interactions are limited. Here, we analyzed 22 single-cell transcriptomes and 36 partially-sequenced genomes of the Arcellinida morphospecies Hyalosphenia papilio, which contains Chlorella algae, to shed light on the amoeba-algae association. By characterizing the genetic diversity of associated Chlorella, we detected two distinct clades that can be linked to host genetic diversity, yet at the same time show a biogeographic signal across sampling sites. Fluorescence and transmission electron microscopy showed the presence of intact algae cells within the amoeba cell. Yet analysis of transcriptome data suggested that the algal nuclei are inactive, implying that instead of a stable, mutualistic relationship, the algae may be temporarily exploited for photosynthetic activity before being digested. Furthermore, differences in gene expression of H. papilio and Hyalosphenia elegans demonstrated increased expression of genes related to oxidative stress. Together, our analyses increase knowledge of this host-symbiont association and reveal 1) higher diversity of associated algae than previously characterized, 2) a transient association between H. papilio and Chlorella with unclear benefits for the algae, 3) algal-induced gene expression changes in the host.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Membrane lipid and expression responses of Saccharolobus islandicus REY15A to acid and cold stress

Archaea adjust the number of cyclopentane rings in their glycerol dibiphytanyl glycerol tetraether (GDGT) membrane lipids as a homeostatic response to environmental stressors such as temperature, pH, and energy availability shifts. However, archaeal expression patterns that correspond with changes in GDGT composition are less understood. Here we characterize the acid and cold stress responses of the thermoacidophilic crenarchaeon Saccharolobus islandicus REY15A using growth rates, core GDGT lipid profiles, transcriptomics and proteomics. We show that both stressors result in impaired growth, lower average GDGT cyclization, and differences in gene and protein expression. Transcription data revealed differential expression of the GDGT ring synthase grsB in response to both acid stress and cold stress. Although the GDGT ring synthase encoded by grsB forms highly cyclized GDGTs with ≥5 ring moieties, S. islandicus grsB upregulation under acidic pH conditions did not correspond with increased abundances of highly cyclized GDGTs. Our observations highlight the inability to predict GDGT changes from transcription data alone. Broader analysis of transcriptomic data revealed that S. islandicus differentially expresses many of the same transcripts in response to both acid and cold stress. These included upregulation of several biosynthetic pathways and downregulation of oxidative phosphorylation and motility. Transcript responses specific to either of the two stressors tested here included upregulation of genes related to proton pumping and molecular turnover in acid stress conditions and upregulation of transposases in cold stress conditions. Overall, our study provides a comprehensive understanding of the GDGT modifications and differential expression characteristic of the acid stress and cold stress responses in S. islandicus.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Signature analysis of high-throughput transcriptomics screening data for mechanistic inference and chemical grouping

Abstract High-throughput transcriptomics (HTTr) uses gene expression profiling to characterize the biological activity of chemicals in in vitro cell-based test systems. As an extension of a previous study testing 44 chemicals, HTTr was used to screen an additional 1,751 unique chemicals from the EPA’s ToxCast collection in MCF7 cells using 8 concentrations and an exposure duration of 6 h. We hypothesized that concentration-response modeling of signature scores could be used to identify putative molecular targets and cluster chemicals with similar bioactivity. Clustering and enrichment analyses were conducted based on signature catalog annotations and ToxPrint chemotypes to facilitate molecular target prediction and grouping of chemicals with similar bioactivity profiles. Enrichment analysis based on signature catalog annotation identified known mechanisms of action (MeOAs) associated with well-studied chemicals and generated putative MeOAs for other active chemicals. Chemicals with predicted MeOAs included those targeting estrogen receptor (ER), glucocorticoid receptor (GR), retinoic acid receptor (RAR), the NRF2/KEAP/ARE pathway, AP-1 activation, and others. Using reference chemicals for ER modulation, the study demonstrated that HTTr in MCF7 cells was able to stratify chemicals in terms of agonist potency, distinguish ER agonists from antagonists, and cluster chemicals with similar activities as predicted by the ToxCast ER Pathway model. Uniform manifold approximation and projection (UMAP) embedding of signature-level results identified novel ER modulators with no ToxCast ER Pathway model predictions. Finally, UMAP combined with ToxPrint chemotype enrichment was used to explore the biological activity of structurally related chemicals. The study demonstrates that HTTr can be used to inform chemical risk assessment by determining in vitro points of departure, predicting chemicals’ MeOA and grouping chemicals with similar bioactivity profiles.

Toxicology↗

DOE OSTI2024

Bayesian Framework for Predicting and Controlling Metabolic Phenotypes in Microbial System

To improve titers, rates and yields for sucrose production in an engineered strain of Synechococcus elongatus PCC7942, we employed Bayesian metabolic control analysis to transcriptomics and external metabolomics data generated for various phases during the circadian clock. Top overexpression candidates included sodium-dependent bicarbonate transporter (H2cO3_Nat_syn), and UTP—glucose-1-phosphate uridylyltransferase (GALUi). Top repression candidates included Glycogen/starch synthetases, ADP-glucose type (GLCS3), Glutamate racemase (GLUR), and ribonucleoside diphosphate reductase (RNDR1).

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Machine learning analysis of RB-TnSeq fitness data predicts functional gene modules in Pseudomonas putida KT2440

ABSTRACT There is growing interest in engineering Pseudomonas putida KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of P. putida relies on the understanding of the functional relationships between genes. In this work, independent component analysis (ICA) was applied to a compendium of existing fitness data from randomly barcoded transposon insertion sequencing (RB-TnSeq) of P. putida KT2440 grown in 179 unique experimental conditions. ICA identified 84 independent groups of genes, which we call fModules (“functional modules”), where gene members displayed shared functional influence in a specific cellular process. This machine learning-based approach both successfully recapitulated previously characterized functional relationships and established hitherto unknown associations between genes. Selected gene members from fModules for hydroxycinnamate metabolism and stress resistance, acetyl coenzyme A assimilation, and nitrogen metabolism were validated with engineered mutants of P. putida . Additionally, functional gene clusters from ICA of RB-TnSeq data sets were compared with regulatory gene clusters from prior ICA of RNAseq data sets to draw connections between gene regulation and function. Because ICA profiles the functional role of several distinct gene networks simultaneously, it can reduce the time required to annotate gene function relative to manual curation of RB-TnSeq data sets. IMPORTANCE This study demonstrates a rapid, automated approach for elucidating functional modules within complex genetic networks. While Pseudomonas putida randomly barcoded transposon insertion sequencing data were used as a proof of concept, this approach is applicable to any organism with existing functional genomics data sets and may serve as a useful tool for many valuable applications, such as guiding metabolic engineering efforts in other microbes or understanding functional relationships between virulence-associated genes in pathogenic microbes. Furthermore, this work demonstrates that comparison of data obtained from independent component analysis of transcriptomics and gene fitness datasets can elucidate regulatory-functional relationships between genes, which may have utility in a variety of applications, such as metabolic modeling, strain engineering, or identification of antimicrobial drug targets.

09 BIOMASS FUELS↗

DOE OSTI2024

Bioenergy sorghum nodal root bud development: morphometric, transcriptomic and gene regulatory network analysis

Bioenergy sorghum’s large and deep nodal root system and associated microbiome enables uptake of water and nutrients from and deposition of soil organic carbon into soil profiles, key contributors to the crop’s resilience and sustainability. The goal of this study was to increase our understanding of bioenergy sorghum nodal root bud development. Sorghum nodal root bud initiation was first observed on the stem node of the 7 th phytomer below the shoot apex. Buds were initiated near the upper end of the stem node pulvinus on the side of the stem opposite the tiller bud, then additional buds were added over the next 6-8 days forming a ring of 10-15 nascent nodal root buds around the stem. Later in plant development, a second ring of nodal root buds began forming on the 17 th stem node immediately above the first ring of buds. Overall, nodal root bud development can take ~40 days from initiation to onset of nodal root outgrowth. Nodal root buds were initiated in close association with vascular bundles in the rind of the pulvinus. Stem tissue forming nascent nodal root buds expressed sorghum homologs of genes associated with root initiation (WOX4), auxin transport (LAX2, PIN4), meristem activation (NGAL2), and genes involved in cell proliferation. Expression of WOX11 and WOX5, genes involved in root stem niche formation, increased early in nodal root bud development followed by genes encoding PLTs, LBDs (LBD29), LRP1, SMB, RGF1 and root cap LEAs later in development. A nodal root bud gene regulatory network module expressed during nodal root bud initiation predicted connections linking PFA5, SPL9 and WOX4 to genes involved in hormone signaling, meristem activation, and cell proliferation. A network module expressed later in development predicted connections among SOMBRERO, a gene involved in root cap formation, and GATA19, BBM, LBD29 and RITF1/RGF1 signaling. Overall, this study provides a detailed description of bioenergy sorghum nodal root bud development and transcriptome information useful for understanding the regulation of sorghum nodal root bud formation and development.

09 BIOMASS FUELS↗

DOE OSTI2021

Identification and selection of optimal reference genes for qPCR-based gene expression analysis in Fucus distichus under various abiotic stresses

Quantitative gene expression analysis is an important tool in the scientist’s belt. The identification of evenly expressed reference genes is necessary for accurate quantitative gene expression analysis, whether by traditional RT-PCR (reverse-transcription polymerase chain reaction) or by qRT-PCR (quantitative real-time PCR; qPCR). In the Stramenopiles (the major line of eukaryotes that includes brown algae) there is a noted lack of known reference genes for such studies, largely due to the absence of available molecular tools. Here we present a set of nine reference genes (Elongation Factor 1 alpha (EF1A), Elongation Factor 2 alpha (EF2A), Elongation Factor 1 beta (EF1B), 14-3-3 Protein, Ubiquitin Conjugating Enzyme (UBCE2), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), Actin Related Protein Complex (ARP2/3), Ribosomal Protein (40s; S23), and Actin) for the brown alga Fucus distichus . These reference genes were tested on adult sporophytes across six abiotic stress conditions (desiccation, light and temperature modification, hormone addition, pollutant exposure, nutrient addition, and wounding). Suitability of these genes as reference genes was quantitatively evaluated across conditions using standard methods and the majority of the tested genes were evaluated favorably. However, we show that normalization genes should be chosen on a condition-by-condition basis. We provide a recommendation that at least two reference genes be used per experiment, a list of recommended pairs for the conditions tested here, and a procedure for identifying a suitable set for an experimenter’s unique design. With the recent expansion of interest in brown algal biology and accompanied molecular tools development, the variety of experimental conditions tested here makes this study a valuable resource for future work in basic biology and understanding stress responses in the brown algal lineage.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Delayed leaf greening involves a major shift in the expression of cytosolic and mitochondrial ribosomes to plastid ribosomes in the highly phosphorus-use-efficient Hakea prostrata (Proteaceae)

Abstract Background and aims Hakea prostrata (Proteaceae) is a highly phosphorus-use-efficient plant native to southwest Australia. It maintains a high photosynthetic rate at low leaf phosphorus (P) and exhibits delayed leaf greening, a convergent adaptation that increases nutrient-use efficiency. This study aimed to provide broad physiological and gene expression profiles across leaf development, uncovering pathways leading from young leaves as nutrient sinks to mature leaves as low-nutrient, energy-transducing sources. Methods To explore gene expression underlying delayed greening, we analysed a de novo transcriptome for H. prostrata across five stages of leaf development. Photosynthesis and respiration rates, and foliar pigment, P and nitrogen (N) concentrations were determined, including the division of P into five biochemical fractions. Key results Transcripts encoding functions associated with leaf structure generally decreased in abundance across leaf development, concomitant with decreases in foliar concentrations of 85% for anthocyanins, 90% for P and 70% for N. The expression of genes associated with photosynthetic function increased during or after leaf expansion, in parallel with increases in photosynthetic pigments and activity, much later in leaf development than in species that do not have delayed greening. As leaves developed, transcript abundance for cytosolic and mitochondrial ribosomal proteins generally declined, whilst transcripts for chloroplast ribosomal proteins increased. Conclusions There was a much longer temporal separation of leaf cell growth from chloroplast development in H. prostrata than is found in species that lack delayed greening. Transcriptome-guided analysis of leaf development in H. prostrata provided insight into delayed greening as a nutrient-saving strategy in severely phosphorus-impoverished landscapes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Advances in mass spectrometry-enabled multiomics at single-cell resolution

We report biological organisms are multifaceted, intricate systems where slight perturbations can result in extensive changes in gene expression, protein abundance and/or activity, and metabolic flux. These changes occur at different timescales, spatially across cells of heterogeneous origins, and within single-cells. Hence, multimodal measurements at the smallest biological scales are necessary to capture dynamic changes in heterogeneous biological systems. Of the analytical techniques used to measure biomolecules, mass spectrometry (MS) has proven to be a powerful option due to its sensitivity, robustness, and flexibility with regard to the breadth of biomolecules that can be analyzed. Recently, many studies have coupled MS to other analytical techniques with the goal of measuring multiple modalities from the same single-cell. It is with these concepts in mind that we focus this review on MS-enabled multiomic measurements at single-cell or near-single- cell resolution.

47 OTHER INSTRUMENTATION↗

DOE OSTI2024

Integrating Chemical Catalysis and Biological Conversion of Carbon Intermediates for Deriving Value-Added Products from Carbon Dioxide

Carbon dioxide valorization represents an appealing approach to reducing greenhouse gases in the atmosphere. While electrocatalysis is an effective tool to reduce CO 2 into small carbon compounds, it becomes increasingly challenging to efficiently produce compounds with more carbon atoms. In contrast, while biological systems struggle to utilize CO 2 , they can readily upcycle other small carbon compounds. This project explores the use of a two-stage process that electrocatalytically converts CO 2 into methanol, formate, or acetate which is subsequently utilized by Methylotuvimicrobium alcaliphilum 20Z to produce medium chain length polyhydroxyalkanoate. A techno-economic analysis and life cycle assessment evaluates the commercial viability of the process as well as its carbon emissions. We show here an enhanced CO 2 -to-methanol electroconversion step coupled with the use of a microbial culture adapted to the process conditions to be the optimal configuration for economic potential.

09 BIOMASS FUELS↗

DOE OSTI2021

Omics-Lethal Human Viruses, Interferon-Stimulated Response Experiment IFNaIHH001

The purpose of this experiment was to evaluate the host interferon-stimulated cellular response to interferon alpha (IFNα) treatment. Sample data was obtained from human immortalized human hepatocyte cells (IHH) treated with or without IFNα for mRNA and miRNA transcriptome expression analysis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Oncogenic Ras and ΔNp63α cooperate to recruit immunosuppressive polymorphonuclear myeloid-derived suppressor cells in a mouse model of squamous cancer pathogenesis

Introduction Amplification of human chromosome 3q26-29, which encodes oncoprotein ΔNp63 among other isoforms of the p63 family, is a feature common to squamous cell carcinomas (SCCs) of multiple tissue origins. Along with overexpression of ΔNp63, activation of the protooncogene, RAS , whether by overexpression or oncogenic mutation, is frequently observed in many cancers. In this study, analysis of transcriptome data from The Cancer Genome Atlas (TCGA) demonstrated that expression of TP63 mRNA , particularly ΔNp63 isoforms, and HRAS are significantly elevated in advanced squamous cell carcinomas of the head and neck (HNSCCs), suggesting pathological significance. However, how co-overexpressed ΔNp63 and HRAS affect the immunosuppressive tumor microenvironment (TME) is incompletely understood. Methods Here, we established and characterized an immune competent mouse model using primary keratinocytes with retroviral-mediated overexpression of ΔNp63α and constitutively activated HRAS (v-ras Ha G12R) to evaluate the role of these oncogenes in the immune TME. Results In this model, orthotopic grafting of wildtype syngeneic keratinocytes expressing both v-ras Ha and elevated levels of ΔNp63α consistently yield carcinomas in syngeneic hosts, while cells expressing v-ras Ha alone yield predominantly papillomas. We found that polymorphonuclear (PMN) myeloid cells, experimentally validated to be immunosuppressive and thus representing myeloid-derived suppressor cells (PMN-MDSCs), were significantly recruited into the TME of carcinomas arising early following orthotopic grafting of ΔNp63α/v-ras Ha -expressing keratinocytes. ΔNp63α/v-ras Ha -driven carcinomas expressed higher levels of chemokines implicated in recruitment of MDSCs compared to v-ras Ha -initiated tumors, providing a heretofore undescribed link between ΔNp63α/HRAS-driven carcinomas and the development of an immunosuppressive TME. Conclusion These results support the utilization of a genetic carcinogenesis model harboring specific genomic drivers of malignancy to study mechanisms underlying the development of local immunosuppression.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Comparative Genomics and Transcriptomics Analyses Reveal Divergent Plant Biomass-Degrading Strategies in Fungi

Plant biomass is one of the most abundant renewable carbon sources, which holds great potential for replacing current fossil-based production of fuels and chemicals. In nature, fungi can efficiently degrade plant polysaccharides by secreting a broad range of carbohydrate-active enzymes (CAZymes), such as cellulases, hemicellulases, and pectinases. Due to the crucial role of plant biomass-degrading (PBD) CAZymes in fungal growth and related biotechnology applications, investigation of their genomic diversity and transcriptional dynamics has attracted increasing attention. In this project, we systematically compared the genome content of PBD CAZymes in six taxonomically distant species, Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens, Trichoderma reesei, Phanerochaete chrysosporium, and Dichomitus squalens, as well as their transcriptome profiles during growth on nine monosaccharides. Considerable genomic variation and remarkable transcriptomic diversity of CAZymes were identified, implying the preferred carbon source of these fungi and their different methods of transcription regulation. In addition, the specific carbon utilization ability inferred from genomics and transcriptomics was compared with fungal growth profiles on corresponding sugars, to improve our understanding of the conversion process. This study enhances our understanding of genomic and transcriptomic diversity of fungal plant polysaccharide-degrading enzymes and provides new insights into designing enzyme mixtures and metabolic engineering of fungi for related industrial applications.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRSDate not supplied

Role of PIEZO1 in T Cell Activation Under Simulated Microgravity

True and simulated microgravity conditions have been well documented to cause the inhibition of T cell activation by mitogens. Although several studies aimed at exploring the mechanisms for such a phenomenon have been published, how this is occurring remains unresolved. PIEZO1 is a known mechanosensing gene and has been shown to be critically involved in human T cell activation. In our analysis of transcriptomics changes in peripheral mononuclear cells (PBMC) collected from the ISS crewmembers in space, the expression of PIEZO1 was downregulated. To investigate the role of the PIEZO1 gene in T cell activation in microgravity, we used rotating wall vessels (RWV), which simulate microgravity on the ground and are known to inhibit T cell activation. In this pilot study, PBMC cultured in RWV and in the static 1g condition were stimulated with Human T-Activator CD3/CD28 beads. The cells were also treated with and without Yoda1, a chemical agonist that activates PIEZO1 independent of mechanical cues or any other cellular component. After culturing for 24 hours, the cells were stained for activation markers and the PIEZO1 antibody, then were analyzed by flow cytometry. Our results indicate reduced T cell activation with mitogen under simulated microgravity, but no changes of the PIEZO1 signals were detected. In addition, the reduced activation was not restored in cells cultured with Yoda1. Taken together, our results suggest that PIEZO1 may play a minimal role in the inhibition of T cell activation in space.

Honglu Wu↗

NASA NTRSDate not supplied

Role of PIEZO1 in T Cell Activation Under Simulated Microgravity

Honglu Wu↗

NASA NTRSDate not supplied

Role of PIEZO1 in T Cell Activation Under Simulated Microgravity

Honglu Wu↗

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 OSTI2023

Genome-wide characterization of the soybean DOMAIN OF UNKNOWN FUNCTION 679 membrane protein gene family highlights their potential involvement in growth and stress response

The DMP (DUF679 membrane proteins) family is a plant-specific gene family that encodes membrane proteins. The DMP family genes are suggested to be involved in various programmed cell death processes and gamete fusion during double fertilization in Arabidopsis. However, their functional relevance in other crops remains unknown. This study identified 14 genes from the DMP family in soybean (Glycine max) and characterized their physiochemical properties, subcellular location, gene structure, and promoter regions using bioinformatics tools. Additionally, their tissue-specific and stress-responsive expressions were analyzed using publicly available transcriptome data. Phylogenetic analysis of 198 DMPs from monocots and dicots revealed six clades, with clade-I encoding senescence-related AtDMP1/2 orthologues and clade-II including pollen-specific AtDMP8/9 orthologues. The largest clade, clade-III, predominantly included monocot DMPs, while monocot- and dicot-specific DMPs were assembled in clade-IV and clade-VI, respectively. Evolutionary analysis suggests that soybean GmDMPs underwent purifying selection during evolution. Using 68 transcriptome datasets, expression profiling revealed expression in diverse tissues and distinct responses to abiotic and biotic stresses. The genes Glyma.09G237500 and Glyma.18G098300 showed pistil-abundant expression by qPCR, suggesting they could be potential targets for female organ-mediated haploid induction. Furthermore, cis-acting regulatory elements primarily related to stress-, hormone-, and light-induced pathways regulate GmDMPs, which is consistent with their divergent expression and suggests involvement in growth and stress responses. Overall, our study provides a comprehensive report on the soybean GmDMP family and a framework for further biological functional analysis of DMP genes in soybean or other crops.

59 BASIC BIOLOGICAL SCIENCES↗