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DOE OSTI2023

Exploring Plant Cis –Regulatory Elements at Single–Cell Resolution: Overcoming Biological and Computational Challenges to Advance Plant Research

Cis-regulatory elements (CREs) are important sequences for gene expression and for plant biological processes such as development, evolution, domestication, and stress response. However, studying CREs in plant genomes has been challenging. The totipotent nature of plant cells, coupled with inability to maintain plant cell types in culture and the inherent technical challenges posed by the cell wall have limited our understanding of how plant cell types acquire and maintain their identities and respond to the environment via CRE usage. Furthermore, advances in single cell epigenomics have revolutionized the field identifying cell-type-specific CREs. These new technologies have the potential to significantly advance our understanding of plant CRE biology, and shed light on how the regulatory genome gives rise to diverse plant phenomena. However, there are significant biological and computational challenges associated with analyzing single cell epigenomic datasets. In this review, we discuss the historical and foundational underpinnings of plant single-cell research, challenges and common pitfalls in analysis of plant single-cell epigenomic data, and highlight biological challenges unique to plants. Additionally, we discuss how the application of single-cell epigenomic data in various contexts stands to transform our understanding of the importance of CREs in plant genomes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

RNAi and genome editing of sugarcane: Progress and prospects

SUMMARY Sugarcane, which provides 80% of global table sugar and 40% of biofuel, presents unique breeding challenges due to its highly polyploid, heterozygous, and frequently aneuploid genome. Significant progress has been made in developing genetic resources, including the recently completed reference genome of the sugarcane cultivar R570 and pan‐genomic resources from sorghum, a closely related diploid species. Biotechnological approaches including RNA interference (RNAi), overexpression of transgenes, and gene editing technologies offer promising avenues for accelerating sugarcane improvement. These methods have successfully targeted genes involved in important traits such as sucrose accumulation, lignin biosynthesis, biomass oil accumulation, and stress response. One of the main transformation methods—biolistic gene transfer or Agrobacterium ‐mediated transformation—coupled with efficient tissue culture protocols, is typically used for implementing these biotechnology approaches. Emerging technologies show promise for overcoming current limitations. The use of morphogenic genes can help address genotype constraints and improve transformation efficiency. Tissue culture‐free technologies, such as spray‐induced gene silencing, virus‐induced gene silencing, or virus‐induced gene editing, offer potential for accelerating functional genomics studies. Additionally, novel approaches including base and prime editing, orthogonal synthetic transcription factors, and synthetic directed evolution present opportunities for enhancing sugarcane traits. These advances collectively aim to improve sugarcane's efficiency as a crop for both sugar and biofuel production. This review aims to discuss the progress made in sugarcane methodologies, with a focus on RNAi and gene editing approaches, how RNAi can be used to inform functional gene targets, and future improvements and applications.

Brant, Eleanor [Agronomy Department, Plant Molecul↗

DOE OSTI2021

Eight Metagenome-Assembled Genomes Provide Evidence for Microbial Adaptation in 20,000- to 1,000,000-Year-Old Siberian Permafrost

Permafrost microbes may be metabolically active in microscopic layers of liquid brines, even in ancient soil. Metagenomics can help discern whether permafrost microbes show adaptations to this environment. Thirty-three metagenome-assembled genomes (MAGs) were obtained from six depths (3.5 m to 20 m) of freshly cored permafrost from the Siberian Kolyma-Indigirka Lowland region. These soils have been continuously frozen for ~20,000 to 1,000,000 years. Eight of these MAGs were ≥80% complete with <10% contamination and were taxonomically identified as Aminicenantes, Atribacteria, Chloroflexi, and Actinobacteria within bacteria and Thermoprofundales within archaea. MAGs from these taxa have been obtained previously from nonpermafrost environments and have been suggested to show adaptations to long-term energy starvation, but they have never been explored in ancient permafrost. The permafrost MAGs had greater proportions in the Clusters of Orthologous Groups (COGs) categories of energy production and conversion and carbohydrate transport and metabolism than did their nonpermafrost counterparts. They also contained genes for trehalose synthesis, thymine metabolism, mevalonate biosynthesis, and cellulose degradation, which were less prevalent in nonpermafrost genomes. Furthermore, many of these genes are involved in membrane stabilization and osmotic stress responses, consistent with adaptation to the anoxic, high-ionic-strength, cold environments of permafrost brine films. Our results suggest that this ancient permafrost contains DNA of high enough quality to assemble MAGs from microorganisms with adaptations to survive long-term freezing in this extreme environment.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2020

Trehalose Degradation by Cellvibrio japonicus Exhibits No Functional Redundancy and Is Solely Dependent on the Tre37A Enzyme

The α-diglucoside trehalose has historically been known as a component of the bacterial stress response, though it more recently has been studied for its relevance in human gut health and biotechnology development. The utilization of trehalose as a nutrient source by bacteria relies on carbohydrate-active enzymes, specifically those of the glycoside hydrolase family 37 (GH37), to degrade the disaccharide into substituent glucose moieties for entry into metabolism. Environmental bacteria using oligosaccharides for nutrients often possess multiple carbohydrate-active enzymes predicted to have the same biochemical activity and therefore are thought to be functionally redundant. In this study, we characterized trehalose degradation by the biotechnologically important saprophytic bacterium Cellvibrio japonicus This bacterium possesses two predicted α-α-trehalase genes, tre37A and tre37B, and our investigation using mutational analysis found that only the former is essential for trehalose utilization by C. japonicus Heterologous expression experiments found that only the expression of the C. japonicus tre37A gene in an Escherichia coli treA mutant strain allowed for full utilization of trehalose. Biochemical characterization of C. japonicus GH37 activity determined that the tre37A gene product is solely responsible for cleaving trehalose and is an acidic α-α-trehalase. Bioinformatic and mutational analyses indicate that Tre37A directly cleaves trehalose to glucose in the periplasm, as C. japonicus does not possess a phosphotransferase system. This study facilitates the development of a comprehensive metabolic model for α-linked disaccharides in C. japonicus and more broadly expands our understanding of the strategies that saprophytic bacteria employ to capture diverse carbohydrates from the environment. The metabolism of trehalose is becoming increasingly important due to the inclusion of this α-diglucoside in a number of foods and its prevalence in the environment. Bacteria able to utilize trehalose in the human gut possess a competitive advantage, as do saprophytic microbes in terrestrial environments. While the biochemical mechanism of trehalose degradation is well understood, what is less clear is how bacteria acquire this metabolite from the environment. The significance of this report is that by using the model saprophyte Cellvibrio japonicus, we were able to functionally characterize the two predicted trehalase enzymes that the bacterium possesses and determined that the two enzymes are not equivalent and are not functionally redundant. Here, the results and approaches used to understand the complex physiology of α-diglucoside metabolism from this study can be applied broadly to other polysaccharide-degrading bacteria.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

The mevalonate pathway of isoprenoid biosynthesis supports metabolic flexibility in Mycobacterium marinum

ABSTRACT Isoprenoids are a diverse class of natural products that are essential in all domains of life. Most bacteria synthesize isoprenoids through either the methylerythritol phosphate (MEP) pathway or the mevalonate (MEV) pathway, while a small subset encodes both pathways, including the pathogen Mycobacterium marinum (Mm). It is unclear whether the MEV pathway is functional in Mm, or why Mm encodes seemingly redundant metabolic pathways. Here, we show that the MEP pathway is essential in Mm, while the MEV pathway is dispensable in culture, with the ΔMEV mutant having no growth defect in axenic culture but a competitive growth defect compared to WT Mm. We found that the MEV pathway does not play a role in ex vivo or in vivo acute infection but does play a role in survival of peroxide stress. Metabolite profiling revealed that modulation of the MEV pathway causes compensatory changes in the concentration of MEP intermediates DOXP and CDP-ME, suggesting that the MEV pathway is functional and that the pathways interact at the metabolic level. Finally, the MEV pathway is upregulated early in the shift down to hypoxia, suggesting that it may provide metabolic flexibility to this bacterium. Interestingly, we found that our complemented strains, which vary in copy number of the polyprenyl synthetase idsB2 , responded differently to peroxide and UV stresses, suggesting a role for this gene as a determinant of downstream prenyl phosphate metabolism. Together, these findings suggest that MEV may serve as an anaplerotic pathway to make isoprenoids under stress conditions. IMPORTANCE Organisms from all domains of life utilize isoprenoids to carry out thousands of critical and auxiliary cellular processes, including signaling, maintaining membrane integrity, stress response, and host-pathogen interactions. The common precursor of all isoprenoids is synthesized via one of two biosynthetic pathways. Importantly, some bacteria encode both pathways, including M. marinum . We found that only one pathway is essential in M. marinum , while the nonessential pathway may confer metabolic flexibility to help the bacterium better adapt to various environmental conditions. We also found that the polyprenyl synthetase IdsB2 plays an important role in driving such phenotypes. Further, we demonstrate metabolic interplay between both functional pathways. These insights represent the first characterization of isoprenoid biosynthesis in dual pathway-encoding mycobacteria.

Qabar, Christine M. [Department of Plant and Micro↗

DOE OSTI2023

Influence of soil depth, irrigation, and plant genotype on the soil microbiome, metaphenome, and carbon chemistry

ABSTRACT Climate change is causing an increase in drought in many soil ecosystems and a loss of soil organic carbon. Calcareous soils may partially mitigate these losses via carbon capture and storage. Here, we aimed to determine how irrigation-supplied soil moisture and perennial plants impact biotic and abiotic soil properties that underpin deep soil carbon chemistry in an unfertilized calcareous soil. Soil was sampled up to 1 m in depth from irrigated and planted field treatments and was analyzed using a suite of omics and chemical analyses. The soil microbial community composition was impacted more by irrigation and plant cover treatments than by soil depth. By contrast, metabolomes, lipidomes, and proteomes differed more with soil depth than treatments. Deep soil (>50 cm) had higher soil pH and calcium concentrations and higher levels of organic acids, bicarbonate, and triacylglycerides. By contrast, surface soil (0–5 cm) had higher concentrations of soil organic matter, organic carbon, oxidizable carbon, and total nitrogen. Surface soils also had higher amounts of sugars, sugar alcohols, phosphocholines, and proteins that reflect osmotic and oxidative stress responses. The lipidome was more responsive to perennial tall wheatgrass treatments compared to the metabolome or proteome, with a striking change in diacylglyceride composition. Permanganate oxidizable carbon was more consistently correlated to metabolites and proteins than soil organic and inorganic carbon and soil organic matter. This study reveals specific compounds that reflect differences in organic, inorganic, and oxidizable soil carbon fractions that are impacted by interactions between irrigation-supplied moisture and plant cover in calcareous soil profiles. IMPORTANCE Carbon is cycled through the air, plants, and belowground environment. Understanding soil carbon cycling in deep soil profiles will be important to mitigate climate change. Soil carbon cycling is impacted by water, plants, and soil microorganisms, in addition to soil mineralogy. Measuring biotic and abiotic soil properties provides a perspective of how soil microorganisms interact with the surrounding chemical environment. This study emphasizes the importance of considering biotic interactions with inorganic and oxidizable soil carbon in addition to total organic carbon in carbonate-containing soils for better informing soil carbon management decisions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Biotic Interactions Are More Important than Propagule Pressure in Microbial Community Invasions

ABSTRACT Microbial probiotics are intended to improve functions in diverse ecosystems, yet probiotics often fail to establish in a preexisting microbiome. This is a species invasion problem. The relative importance of the two major factors controlling establishment in this context—propagule pressure (inoculation dose and frequency) and biotic interactions (composition of introduced and resident communities)—is unknown. We tested the effect of these factors in driving microbial composition and functioning following 12 microbial community invasions (e.g., introductions of many microbial invaders) in microcosms. Ecosystem functioning over a 30-day postinvasion period was assessed by measuring activity (respiration) and environment modification (dissolved organic carbon abundance). To test the dependence on environmental context, experiments were performed in two resource environments. In both environments, biotic interactions were more important than propagule pressure in driving microbial composition and community function, but the magnitude of effect varied by environment. Successful invaders comprised approximately 8% of the total number of operational taxonomic units (OTUs). Bacteria were better invaders than fungi, with average relative abundances of 7.4% ± 6.8% and 1.5% ± 1.4% of OTUs, respectively. Common bacterial invaders were associated with stress response traits. The most resilient bacterial and fungal families, in other words, those least impacted by invasions, were linked to antimicrobial resistance or production traits. Illuminating the principles that determine community composition and functioning following microbial invasions is key to efficient community engineering. IMPORTANCE With increasing frequency, humans are introducing new microbes into preexisting microbiomes to alter functioning. Example applications include modification of microflora in human guts for better health and those of soil for food security and/or climate management. Probiotic applications are often approached as trial-and-error endeavors and have mixed outcomes. We propose that increased success in microbiome engineering may be achieved with a better understanding of microbial invasions. We conducted a microbial community invasion experiment to test the relative importance of propagule pressure and biotic interactions in driving microbial community composition and ecosystem functioning in microcosms. We found that biotic interactions were more important than propagule pressure in determining the impact of microbial invasions. Furthermore, the principles for community engineering vary among organismal groups (bacteria versus fungi).

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

ENSO Dynamics in the E3SM-1-0, CESM2, and GFDL-CM4 Climate Models

We report that this study examines historical simulations of ENSO in the E3SM-1-0, CESM2, and GFDL-CM4 climate models, provided by three leading U.S. modeling centers as part of the Coupled Model Intercomparison Project phase 6 (CMIP6). These new models have made substantial progress in simulating ENSO’s key features, including: amplitude; timescale; spatial patterns; phase-locking; spring persistence barrier; and recharge oscillator dynamics. However, some important features of ENSO are still a challenge to simulate. In the central and eastern equatorial Pacific, the models’ weaker-than-observed subsurface zonal current anomalies and zonal temperature gradient anomalies serve to weaken the nonlinear zonal advection of subsurface temperatures, leading to insufficient warm/cold asymmetry of ENSO’s sea surface temperature anomalies (SSTA). In the western equatorial Pacific, the models’ excessive simulated zonal SST gradients amplify their zonal temperature advection, causing their SSTA to extend farther west than observed. The models underestimate both ENSO’s positive dynamic feedbacks (due to insufficient zonal wind stress responses to SSTA) and its thermodynamic damping (due to insufficient convective cloud shading of eastern Pacific SSTA during warm events); compensation between these biases leads to realistic linear growth rates for ENSO, but for somewhat unrealistic reasons. The models also exhibit stronger-than-observed feedbacks onto eastern equatorial Pacific SSTAs from thermocline depth anomalies, which accelerates the transitions between events and shortens the simulated ENSO period relative to observations. Implications for diagnosing and simulating ENSO in climate models are discussed.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2025

Plasma proteomic biomarkers of physical frailty in heart failure: a propensity score matched discovery-based pilot study

Background: Physical frailty is highly prevalent in heart failure (HF), but we lack an understanding of the underlying pathophysiology. Proteomics evaluation of plasma samples may elucidate potential mechanisms and biomarkers of physical frailty in HF. We aimed to identify plasma proteomic biomarkers that are differentially expressed between physically frail and non physically frail adults with HF. Methods: This was a secondary analysis of a subset of data and plasma samples from a study of frailty among patients with New York Heart Association (NYHA) Functional Classification I-IV HF. Physical frailty was measured using the Frailty Phenotype Criteria. Propensity score matching was used to match pairs of physically frail (n = 20) vs. non-physically frail (n = 20) patients on clinical characteristics. Plasma samples were processed using a sensitive liquid chromatography mass spectrometry platform, utilizing a multiplexed tandem mass tag-labeled quantitative proteomics approach. Differentially expressed proteins were quantified individually using paired t tests with associated log fold change of 0.3 and Fisher’s combined p values. Results: The sample (n = 40) was 62.8±16.9 years old, 58% female, and 55% NYHA Class III/IV. Proteomics analysis revealed 7 proteins differentially expressed using full differential criteria: matrix metalloproteinase-14 was downregulated in frailty, and copine-1, low affinity immunoglobulin gamma Fc region receptor III-A and III-B, probable non-functional immunoglobulin kappa variable 2D-24, glutathione S-transferase Mu 1, and argininosuccinate lyase were upregulated in frailty. Conclusions: Proteomic biomarkers related to the immune system, stress response, and detoxification were differentially expressed between physically frail and non-physically frail adults with HF.

Biomarkers↗

DOE OSTI2021

Parsed synthesis of pyocyanin via co-culture enables context-dependent intercellular redox communication

Abstract Background Microbial co-cultures and consortia are of interest in cell-based molecular production and even as “smart” therapeutics in that one can take advantage of division of labor and specialization to expand both the range of available functions and mechanisms for control. The development of tools that enable coordination and modulation of consortia will be crucial for future application of multi-population cultures. In particular, these systems would benefit from an expanded toolset that enables orthogonal inter-strain communication. Results We created a co-culture for the synthesis of a redox-active phenazine signaling molecule, pyocyanin (PYO), by dividing its synthesis into the generation of its intermediate, phenazine carboxylic acid (PCA) from the first strain, followed by consumption of PCA and generation of PYO in a second strain. Interestingly, both PCA and PYO can be used to actuate gene expression in cells engineered with the soxRS oxidative stress regulon, although importantly this signaling activity was found to depend on growth media. That is, like other signaling motifs in bacterial systems, the signaling activity is context dependent. We then used this co-culture’s phenazine signals in a tri-culture to modulate gene expression and production of three model products: quorum sensing molecule autoinducer-1 and two fluorescent marker proteins, eGFP and DsRed. We also showed how these redox-based signals could be intermingled with other quorum-sensing (QS) signals which are more commonly used in synthetic biology, to control complex behaviors. To provide control over product synthesis in the tri-cultures, we also showed how a QS-induced growth control module could guide metabolic flux in one population and at the same time guide overall tri-culture function. Specifically, we showed that phenazine signal recognition, enabled through the oxidative stress response regulon soxRS, was dependent on media composition such that signal propagation within our parsed synthetic system could guide different desired outcomes based on the prevailing environment. In doing so, we expanded the range of signaling molecules available for coordination and the modes by which they can be utilized to influence overall function of a multi-population culture. Conclusions Our results show that redox-based signaling can be intermingled with other quorum sensing signaling in ways that enable user-defined control of microbial consortia yielding various outcomes defined by culture medium. Further, we demonstrated the utility of our previously designed growth control module in influencing signal propagation and metabolic activity is unimpeded by orthogonal redox-based signaling. By exploring novel multi-modal strategies for guiding communication and consortia outcome, the concepts introduced here may prove to be useful for coordination of multiple populations within complex microbial systems.

Chun, Kayla↗

DOE OSTI2022

Adapted laboratory evolution of Thermotoga sp. strain RQ7 under carbon starvation

Abstract Objective Adaptive laboratory evolution (ALE) is an effective approach to study the evolution behavior of bacterial cultures and to select for strains with desired metabolic features. In this study, we explored the possibility of evolving Thermotoga sp. strain RQ7 for cellulose-degrading abilities. Results Wild type RQ7 strain was subject to a series of transfers over six and half years with cellulose filter paper as the main and eventually the sole carbon source. Each transfer was accompanied with the addition of 50 μg of Caldicellulosiruptor saccharolyticus DSM 8903 genomic DNA. A total of 331 transfers were completed. No cellulose degradation was observed with the RQ7 cultures. Thirty three (33) isolates from six time points were sampled and sequenced. Nineteen (19) of the 33 isolates were unique, and the rest were duplicated clones. None of the isolates acquired C. saccharolyticus DNA, but all accumulated small-scale mutations throughout their genomes. Sequence analyses revealed 35 mutations that were preserved throughout the generations and another 15 mutations emerged near the end of the study. Many of the affected genes participate in phosphate metabolism, substrate transport, stress response, sensory transduction, and gene regulation.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2020

Small and mighty: adaptation of superphylum Patescibacteria to groundwater environment drives their genome simplicity

The newly defined superphylum Patescibacteria such as Parcubacteria (OD1) and Microgenomates (OP11) has been found to be prevalent in groundwater, sediment, lake, and other aquifer environments. Recently increasing attention has been paid to this diverse superphylum including > 20 candidate phyla (a large part of the candidate phylum radiation, CPR) because it refreshed our view of the tree of life. However, adaptive traits contributing to its prevalence are still not well known. Results Here, we investigated the genomic features and metabolic pathways of Patescibacteria in groundwater through genome-resolved metagenomics analysis of >600 Gbp sequence data. We observed that, while the members of Patescibacteria have reduced genomes (~ 1 Mbp) exclusively, functions essential to growth and reproduction such as genetic information processing were retained. Surprisingly, they have sharply reduced redundant and nonessential functions, including specific metabolic activities and stress response systems. The Patescibacteria have ultra-small cells and simplified membrane structures, including flagellar assembly, transporters, and two-component systems. Despite the lack of CRISPR viral defense, the bacteria may evade predation through deletion of common membrane phage receptors and other alternative strategies, which may explain the low representation of prophage proteins in their genomes and lack of CRISPR. By establishing the linkages between bacterial features and the groundwater environmental conditions, our results provide important insights into the functions and evolution of this CPR group.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Metagenomic insights into the taxonomy, function, and dysbiosis of prokaryotic communities in octocorals

Background. In octocorals (Cnidaria Octocorallia), the functional relationship between host health and its symbiotic consortium has yet to be determined. Here, we employed comparative metagenomics to uncover the distinct functional and phylogenetic features of the microbiomes of healthy Eunicella gazella, Eunicella verrucosa, and Leptogorgia sarmentosa tissues, in contrast with the microbiomes found in seawater and sediments. We further explored how the octocoral microbiome shifts to a pathobiome state in E. gazella. Results. Multivariate analyses based on 16S rRNA genes, Clusters of Orthologous Groups of proteins (COGs), Protein families (Pfams), and secondary metabolite-biosynthetic gene clusters annotated from 20 Illumina-sequenced metagenomes each revealed separate clustering of the prokaryotic communities of healthy tissue samples of the three octocoral species from those of necrotic E. gazella tissue and surrounding environments. While the healthy octocoral microbiome was distinguished by so-far uncultivated Endozoicomonadaceae, Oceanospirillales, and Alteromonadales phylotypes in all host species, a pronounced increase of Flavobacteriaceae and Alphaproteobacteria, originating from seawater, was observed in necrotic E. gazella tissue. Increased abundances of eukaryotic-like proteins, exonucleases, restriction endonucleases, CRISPR/Cas proteins, and genes encoding for heat-shock proteins, inorganic ion transport, and iron storage distinguished the prokaryotic communities of healthy octocoral tissue regardless of the host species. An increase of arginase and nitric oxide reductase genes, observed in necrotic E. gazella tissues, suggests the existence of a mechanism for suppression of nitrite oxide production by which octocoral pathogens may overcome the host’s immune system. Conclusions. This is the first study to employ primer-less, shotgun metagenome sequencing to unveil the taxonomic, functional, and secondary metabolism features of prokaryotic communities in octocorals. Our analyses reveal that the octocoral microbiome is distinct from those of the environmental surroundings, is host genus (but not species) specific, and undergoes large, complex structural changes in the transition to the dysbiotic state. Host-symbiont recognition, abiotic-stress response, micronutrient acquisition, and an antiviral defense arsenal comprising multiple restriction endonucleases, CRISPR/Cas systems, and phage lysogenization regulators are signatures of prokaryotic communities in octocorals. We argue that these features collectively contribute to the stabilization of symbiosis in the octocoral holobiont and constitute beneficial traits that can guide future studies on coral reef conservation and microbiome therapy.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

PKA regulatory subunit Bcy1 couples growth, lipid metabolism, and fermentation during anaerobic xylose growth in Saccharomyces cerevisiae

Organisms have evolved elaborate physiological pathways that regulate growth, proliferation, metabolism, and stress response. These pathways must be properly coordinated to elicit the appropriate response to an ever-changing environment. While individual pathways have been well studied in a variety of model systems, there remains much to uncover about how pathways are integrated to produce systemic changes in a cell, especially in dynamic conditions. We previously showed that deletion of Protein Kinase A (PKA) regulatory subunit BCY1 can decouple growth and metabolism in Saccharomyces cerevisiae engineered for anaerobic xylose fermentation, allowing for robust fermentation in the absence of division. This provides an opportunity to understand how PKA signaling normally coordinates these processes. Here, we integrated transcriptomic, lipidomic, and phospho-proteomic responses upon a glucose to xylose shift across a series of strains with different genetic mutations promoting either coupled or decoupled xylose-dependent growth and metabolism. Together, results suggested that defects in lipid homeostasis limit growth in the bcy1Δ strain despite robust metabolism. To further understand this mechanism, we performed adaptive laboratory evolutions to re-evolve coupled growth and metabolism in the bcy1Δ parental strain. The evolved strain harbored mutations in PKA subunit TPK1 and lipid regulator OPI1 , among other genes, and evolved changes in lipid profiles and gene expression. Deletion of the evolved opi1 gene partially reverted the strain’s phenotype to the bcy1Δ parent, with reduced growth and robust xylose fermentation. We suggest several models for how cells coordinate growth, metabolism, and other responses in budding yeast and how restructuring these processes enables anaerobic xylose utilization.

59 BASIC BIOLOGICAL SCIENCES↗

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 OSTI2022

Neutronics Analysis in Preparation of the Blue Room Experiment

The goal of the Blue Room experiment is to investigate the ‘thermal shock’ stress in a tungsten target provoked by proton pulse impact. Short, strong pulses of highly energetic protons cause a rapid energy deposition in tungsten, which leads to a sudden temperature rise. This thermal shock causes a stress response and leads to fatigue, which can limit the lifetime of the target. In the Blue Room experiment, tungsten blocks will be irradiated by pulses of 800 MeV protons with several intensities to gain understanding in the strain response of the tungsten block.

36 MATERIALS SCIENCE↗

DOE OSTI2024

Conserved genetic mechanisms for biotic stress in sorghum (Final report)

Developing durable disease resistance for biofuel crops is crucial, particularly as the range of biofuel crop production expands and pathogens of other plant species evolve to cause diseases of bioenergy feedstocks. Setosphaeria species are significant pathogens of the Andropogonae, and S. turcica can infect both maize and sorghum. Sorghum leaf blight (SLB), caused by S. turcica, is widespread and can decrease grain yields up to 50%, reduce forage quantity and quality, and predispose plants to other diseases, such as anthracnose. Our overall objective was to gain a systems-level understanding of the pathosystem by leveraging natural genetic variation, host specificity of the pathogen, and transcriptome analysis to improve biotic stress resistance in sorghum. We proposed three specific goals: 1) Identify sorghum alleles conferring resistance to S. turcica using natural genetic variation. 2) Characterize genes associated with biotic stress response in sorghum. 3) Identify fungal genes responsible for host specificity.

09 BIOMASS FUELS↗

DOE OSTI2024

Conserved genetic mechanisms for biotic stress in sorghum (Final Report)

09 BIOMASS FUELS↗