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At least 1,621 records · Page 90
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Insights into regulatory T-cell and type-I interferon roles in determining abacavir-induced hypersensitivity or immune tolerance

Introduction Clinical use of several small molecule drugs may lead to severe T-cell-mediated idiosyncratic drug hypersensitivity reactions (iDHR) linked to HLA alleles, including abacavir (ABC) with HLA-B*57:01. Due to study limitations in humans, pathogenic networks in iDHR remain elusive. HLA transgenic murine models have been proposed to bridge knowledge gaps in tolerance and susceptibility to drugs. Methods Mice expressing HLA-B*57:01 and Foxp3-DTR/EGFP were generated to selectively deplete regulatory T-cells (Treg) with diphtheria toxin. ABC was administered for 8 days alone or together with cell- and cytokine-depleting antibodies. Cellular and transcriptomic responses were analyzed by RNA, flow cytometry and fluorescence methods. Results While CD8 + T-cell responses to ABC require HLA presentation, ABC also triggered mitochondrial stress in macrophagesin vitro, independently of HLA.In vivo, Treg were the primary mechanism of drug tolerance controlling HLA presentation and costimulation by antigen presenting cells. Treg ablation uncovered immune adverse events linked to activation and proliferation of both drug-specific and bystander CD8 + T-cells through CD28-mediated pathways with support from CD4 + non-Treg. Type-I interferon (IFN-I) and cellular-stress pathways influenced the fate of lymph node cells responding to ABC, implicating innate immune cells such as macrophages and plasmacytoid dendritic cells in the development of T-cell responses against the drug. IFN-I and IL-2 were necessary for CD8 + T-cell differentiation and ABC-induced adverse reactions. Conclusions This study unveils novel immune mechanisms driven by drug and host-related factors required forin vivoreactions and sheds light on potential biomarker and therapeutic targets for managing and preventing severe and life-threatening iDHR.

Immunology↗
DOE OSTI2025

Feed status and skin injury modulate immunopathology, global gene expression, and survival in channel catfish during virulent Aeromonas hydrophila infection

Introduction VirulentAeromonas hydrophilais a major pathogen in channel catfish (Ictalurus punctatus), that causes motileAeromonassepticemia and significant economic losses. We investigated the effect of feeding status and skin integrity on the host immune response, disease survival, and gastrointestinal pathology following a vAh challenge. Methods Using a bath immersion model, channel catfish were divided into four treatment groups: fin clipped and fed (FCF), fin clipped but not fed (FCN), not fin clipped but fed (NCF), and not fin clipped nor fed (NCN) alongside non-challenged control groups The FCF and NCF groups were fed 2 h prior to the challenge, but the FCN and NCN groups were not. Survival analysis, histopathological assessment, and RNA sequencing were conducted across groups at different time intervals throughout the vAh challenge. Results Survival rates were lowest in the FCF and FCN groups (30% and 23% survival, respectively), suggesting that both feeding and skin damage contributed to disease severity. Histopathological analyses revealed more severe intestinal and gastric lesions in fed groups, characterized by epithelial necrosis, hemorrhage, and edema. Transcriptomic analysis among the groups identified significant differentially expressed genes associated with inflammation, apoptosis, and metabolic stress, with notable upregulation of interleukin 1-beta (il-1β), and complement C3 (c3). Gene ontology enrichment highlighted distinct immune activation patterns between fed and unfed groups, with enhanced pathogen recognition and pro-inflammatory responses in unfed fish. Discussion These findings suggest feeding prior to infection may exacerbate disease pathology, potentially by creating a physiological state conducive to facilitate pathogen proliferation and dampened early immune responses, whereas short-term fasting appears to promote early immune activation. This study provides novel insights into the complex interplay between feed status, physical injury, and immune response to vAh infection.

Immunology↗
DOE OSTI2013

Transcriptional regulation of the carbohydrate utilization network in Thermotoga maritima

Hyperthermophilic bacteria from the Thermotogales lineage can produce hydrogen by fermenting a wide range of carbohydrates. Previous experimental studies identified a large fraction of genes committed to carbohydrate degradation and utilization in the model bacterium Thermotoga maritima. Knowledge of these genes enabled comprehensive reconstruction of biochemical pathways comprising the carbohydrate utilization network. However, transcriptional factors (TFs) and regulatory mechanisms driving this network remained largely unknown. Here, we used an integrated approach based on comparative analysis of genomic and transcriptomic data for the reconstruction of the carbohydrate utilization regulatory networks in 11 Thermotogales genomes. We identified DNA-binding motifs and regulons for 19 orthologous TFs in the Thermotogales. The inferred regulatory network in T. maritima contains 181 genes encoding TFs, sugar catabolic enzymes and ABC-family transporters. In contrast to many previously described bacteria, a transcriptional regulation strategy of Thermotoga does not employ global regulatory factors. The reconstructed regulatory network in T. maritima was validated by gene expression profiling on a panel of mono- and disaccharides and by in vitro DNA-binding assays. The observed upregulation of genes involved in catabolism of pectin, trehalose, cellobiose, arabinose, rhamnose, xylose, glucose, galactose, and ribose showed a strong correlation with the UxaR, TreR, BglR, CelR, AraR, RhaR, XylR, GluR, GalR, and RbsR regulons. Ultimately, this study elucidated the transcriptional regulatory network and mechanisms controlling expression of carbohydrate utilization genes in T. maritima. In addition to improving the functional annotations of associated transporters and catabolic enzymes, this research provides novel insights into the evolution of regulatory networks in Thermotogales.

Microbiology↗
DOE OSTI2014

CO2 exposure at pressure impacts metabolism and stress responses in the model sulfate-reducing bacterium Desulfovibrio vulgaris strain Hildenborough

Geologic carbon dioxide (CO2) sequestration drives physical and geochemical changes in deep subsurface environments that impact indigenous microbial activities. The combined effects of pressurized CO2 on a model sulfate-reducing microorganism, Desulfovibrio vulgaris, have been assessed using a suite of genomic and kinetic measurements. Novel high-pressure NMR time-series measurements using 13C-lactate were used to track D. vulgaris metabolism. We identified cessation of respiration at CO2 pressures of 10 bar, 25 bar, 50 bar, and 80 bar. Concurrent experiments using N2 as the pressurizing phase had no negative effect on microbial respiration, as inferred from reduction of sulfate to sulfide. Complementary pressurized batch incubations and fluorescence microscopy measurements supported NMR observations, and indicated that non-respiring cells were mostly viable at 50 bar CO2 for at least four hours, and at 80 bar CO2 for two hours. The fraction of dead cells increased rapidly after four hours at 80 bar CO2. Transcriptomic (RNA-Seq) measurements on mRNA transcripts from CO2-incubated biomass indicated that cells up-regulated the production of certain amino acids (leucine, isoleucine) following CO2 exposure at elevated pressures, likely as part of a general stress response. Evidence for other poorly understood stress responses were also identified within RNA-Seq data, suggesting that while pressurized CO2 severely limits the growth and respiration of D. vulgaris cells, biomass retains intact cell membranes at pressures up to 80 bar CO2. Together, these data show that geologic sequestration of CO2 may have significant impacts on rates of sulfate reduction in many deep subsurface environments where this metabolism is a key respiratory process.

Desulfovibrio↗
DOE OSTI2016

Experimental Identification of Small Non-Coding RNAs in the Model Marine Bacterium Ruegeria pomeroyi DSS-3

In oligotrophic ocean waters where bacteria are often subjected to chronic nutrient limitation, community transcriptome sequencing has pointed to the presence of highly abundant small RNAs (sRNAs). The role of sRNAs in regulating response to nutrient stress was investigated in a model heterotrophic marine bacterium Ruegeria pomeroyi grown in continuous culture under carbon (C) and nitrogen (N) limitation. RNAseq analysis identified 99 putative sRNAs. Sixty-nine were cis-encoded and located antisense to a presumed target gene. Thirty were trans-encoded and initial target prediction was performed computationally. The most prevalent functional roles of genes anti-sense to the cis-sRNAs were transport, cell-cell interactions, signal transduction, and transcriptional regulation. Most sRNAs were transcribed equally under both C and N limitation, and may be involved in a general stress response. However, 14 were regulated differentially between the C and N treatments and may respond to specific nutrient limitations. A network analysis of the predicted target genes of the R. pomeroyi cis-sRNAs indicated that they average fewer connections than typical protein-encoding genes, and appear to be more important in peripheral or niche-defining functions encoded in the pan genome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

A Complex Endomembrane System in the Archaeon Ignicoccus hospitalis Tapped by Nanoarchaeum equitans

Based on serial sectioning, focused ion beam scanning electron microscopy (FIB/SEM), and electron tomography, we depict in detail the highly unusual anatomy of the marine hyperthermophilic crenarchaeon, Ignicoccus hospitalis. Our data support a complex and dynamic endomembrane system consisting of cytoplasmic protrusions, and with secretory function. Moreover, we reveal that the cytoplasm of the putative archaeal ectoparasite Nanoarchaeum equitans can get in direct contact with this endomembrane system, complementing and explaining recent proteomic, transcriptomic and metabolomic data on this inter-archaeal relationship. In addition, we identified a matrix of filamentous structures and/or tethers in the voluminous inter-membrane compartment (IMC) of I. hospitalis, which might be responsible for membrane dynamics. Overall, this unusual cellular compartmentalization, ultrastructure and dynamics in an archaeon that belongs to the recently proposed TACK superphylum prompts speculation that the eukaryotic endomembrane system might originate from Archaea.

3D↗
DOE OSTI2018

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors

Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species ( Colletotrichum acutatum , Colletotrichum gloesporioides , Fusarium oxysporum , nectria hematococca , and Botrosphaeria dothidea ) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector-based classification was found to be highly consistent with the phylogenomic trees. Identification of lineage-specific effectors is a key step toward understanding C. cassiicola virulence and host specialization mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Rare Earth Elements Alter Redox Balance in Methylomicrobium alcaliphilum 20Z R

Background: Rare Earth Elements (REEs) control methanol utilization in both methane- and methanol-utilizing microbes. It has been established that the addition of REEs leads to the transcriptional repression of MxaFI-MeDH (a two-subunit methanol dehydrogenase, calcium-dependent) and the activation of XoxF-MeDH (a one-subunit methanol dehydrogenase, lanthanumdependent). Both enzymes are pyrroquinoline quinone-dependent alcohol dehydrogenases and show significant homology; however, they display different kinetic properties and substrate specificities. This study investigates the impact of the MxaFI to XoxF switch on the behavior of metabolic networks at a global scale. Results: In this study we investigated the steady-state growth of Methylomicrobium alcaliphilum 20Z R in media containing calcium (Ca) or lanthanum (La, a REE element). We found that cells supplemented with La show a higher growth rate compared to Ca-cultures; however, the efficiency of carbon conversion, estimated as biomass yield, is higher in cells grown with Ca. Three complementary global-omics approaches—RNA-seq transcriptomics, proteomics, and metabolomics—were applied to investigate the mechanisms of improved growth vs carbon conversion. Cells grown with La showed the transcriptional activation of the xoxF gene, a homologue of the formaldehyde-activating enzyme (fae2), a putative transporter, genes for hemin-transport proteins, and nitrate reductase. In contrast, genes for mxaFI and associated cytochrome (mxaG) expression were downregulated. Proteomic profiling suggested additional adjustments of the metabolic network at the protein level, including carbon assimilation pathways, electron transport systems, and the tricarboxylic acid (TCA) cycle. Discord between gene expression and protein abundance changes points toward the possibility of post-transcriptional control of the related systems including key enzymes of the TCA cycle and a set of electron-transport carriers. Metabolomic data followed proteomics and showed the reduction of the ribulosemonophosphate (RuMP) pathway intermediates and the increase of the TCA cycle metabolites. Conclusions: Cells exposed to REEs display higher rates of growth but have lower carbon conversion efficiency compared to cells supplemented with Ca. The most plausible explanation for these physiological changes is an increased conversion of methanol into formate by XoxF-MeDH, which further stimulates methane oxidation but limits both the supply of reducing power and flux of formaldehyde into the RuMP pathway.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Investigating Abiotic and Biotic Mechanisms of Pyrite Reduction

Pyrite (FeS 2 ) has a very low solubility and therefore has historically been considered a sink for iron (Fe) and sulfur (S) and unavailable to biology in the absence of oxygen and oxidative weathering. Anaerobic methanogens were recently shown to reduce FeS 2 and assimilate Fe and S reduction products to meet nutrient demands. However, the mechanism of FeS 2 mineral reduction and the forms of Fe and S assimilated by methanogens remained unclear. Thermodynamic calculations described herein indicate that H 2 at aqueous concentrations as low as 10 –10 M favors the reduction of FeS 2 , with sulfide (HS – ) and pyrrhotite (Fe 1–x S) as products; abiotic laboratory experiments confirmed the reduction of FeS 2 with dissolved H 2 concentrations greater than 1.98 × 10 –4 M H 2 . Growth studies of Methanosarcina barkeri provided with FeS 2 as the sole source of Fe and S resulted in H 2 production but at concentrations too low to drive abiotic FeS 2 reduction, based on abiotic laboratory experimental data. A strain of M. barkeri with deletions in all [NiFe]-hydrogenases maintained the ability to reduce FeS 2 during growth, providing further evidence that extracellular electron transport (EET) to FeS 2 does not involve H 2 or [NiFe]-hydrogenases. Physical contact between cells and FeS 2 was required for mineral reduction but was not required to obtain Fe and S from dissolution products. The addition of a synthetic electron shuttle, anthraquinone-2,6-disulfonate, allowed for biological reduction of FeS 2 when physical contact between cells and FeS 2 was prohibited, indicating that exogenous electron shuttles can mediate FeS 2 reduction. Here, transcriptomics experiments revealed upregulation of several cytoplasmic oxidoreductases during growth of M. barkeri on FeS 2 , which may indicate involvement in provisioning low potential electrons for EET to FeS 2 . Collectively, the data presented herein indicate that reduction of insoluble FeS 2 by M. barkeri occurred via electron transfer from the cell surface to the mineral surface resulting in the generation of soluble HS – and mineral-associated Fe 1–x S. Solubilized Fe(II), but not HS – , from mineral-associated Fe 1–x S reacts with aqueous HS – yielding aqueous iron sulfur clusters (FeS aq ) that likely serve as the Fe and S source for methanogen growth and activity. FeS aq nucleation and subsequent precipitation on the surface of cells may result in accelerated EET to FeS 2 , resulting in positive feedback between cell activity and FeS 2 reduction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

An EvoDevo Study of Salmonid Visual Opsin Dynamics and Photopigment Spectral Sensitivity

Salmonids are ideal models as many species follow a distinct developmental program from demersal eggs and a large yolk sac to hatching at an advanced developmental stage. Further, these economically important teleosts inhabit both marine- and freshwaters and experience diverse light environments during their life histories. At a genome level, salmonids have undergone a salmonid-specific fourth whole genome duplication event (Ss4R) compared to other teleosts that are already more genetically diverse compared to many non-teleost vertebrates. Thus, salmonids display phenotypically plastic visual systems that appear to be closely related to their anadromous migration patterns. This is most likely due to a complex interplay between their larger, more gene-rich genomes and broad spectrally enriched habitats; however, the molecular basis and functional consequences for such diversity is not fully understood. This study used advances in genome sequencing to identify the repertoire and genome organization of visual opsin genes (those primarily expressed in retinal photoreceptors) from six different salmonids [Atlantic salmon ( Salmo salar ), brown trout ( Salmo trutta ), Chinook salmon ( Oncorhynchus tshawytcha ), coho salmon ( Oncorhynchus kisutch ), rainbow trout ( Oncorhynchus mykiss ), and sockeye salmon ( Oncorhynchus nerka )] compared to the northern pike ( Esox lucius ), a closely related non-salmonid species. Results identified multiple orthologues for all five visual opsin classes, except for presence of a single short-wavelength-sensitive-2 opsin gene. Several visual opsin genes were not retained after the Ss4R duplication event, which is consistent with the concept of salmonid rediploidization. Developmentally, transcriptomic analyzes of Atlantic salmon revealed differential expression within each opsin class, with two of the long-wavelength-sensitive opsins not being expressed before first feeding. Also, early opsin expression in the retina was located centrally, expanding dorsally and ventrally as eye development progressed, with rod opsin being the dominant visual opsin post-hatching. Modeling by spectral tuning analysis and atomistic molecular simulation, predicted the greatest variation in the spectral peak of absorbance to be within the Rh2 class, with a ∼40 nm difference in λ max values between the four medium-wavelength-sensitive photopigments. Overall, it appears that opsin duplication and expression, and their respective spectral tuning profiles, evolved to maximize specialist color vision throughout an anadromous lifecycle, with some visual opsin genes being lost to tailor marine-based vision.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

The importance of living botanical collections for plant biology and the “next generation” of evo-devo research

Living botanical collections include germplasm repositories, long-term experimental plantings, and botanical gardens. We present here a series of vignettes to illustrate the central role that living collections have played in plant biology research, including evo-devo research. Looking toward the future, living collections will become increasingly important in support of future evo-devo research. The driving force behind this trend is nucleic acid sequencing technologies, which are rapidly becoming more powerful and cost-effective, and which can be applied to virtually any species. This allows for more extensive sampling, including non-model organisms with unique biological features and plants from diverse phylogenetic positions. Importantly, a major challenge for sequencing-based evo-devo research is to identify, access, and propagate appropriate plant materials. We use a vignette of the ongoing 1,000 Transcriptomes project as an example of the challenges faced by such projects. We conclude by identifying some of the pinch points likely to be encountered by future evo-devo researchers, and how living collections can help address them.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2013

Fermentation metabolism and its evolution in algae

Fermentation or anoxic metabolism allows unicellular organisms to colonize environments that become anoxic. Free-living unicellular algae capable of a photoautotrophic lifestyle can also use a range of metabolic circuitry associated with different branches of fermentation metabolism. While algae that perform mixed-acid fermentation are widespread, the use of anaerobic respiration is more typical of eukaryotic heterotrophs. The occurrence of a core set of fermentation pathways among the algae provides insights into the evolutionary origins of these pathways, which were likely derived from a common ancestral eukaryote. Based on genomic, transcriptomic, and biochemical studies, anaerobic energy metabolism has been examined in more detail in Chlamydomonas reinhardtii (Chlamydomonas) than in any other photosynthetic protist. This green alga is metabolically flexible and can sustain energy generation and maintain cellular redox balance under a variety of different environmental conditions. Fermentation metabolism in Chlamydomonas appears to be highly controlled, and the flexible use of the different branches of fermentation metabolism has been demonstrated in studies of various metabolic mutants. Additionally, when Chlamydomonas ferments polysaccharides, it has the ability to eliminate part of the reductant (to sustain glycolysis) through the production of H 2 , a molecule that can be developed as a source of renewable energy.To date, little is known about the specific role(s) of the different branches of fermentation metabolism, how photosynthetic eukaryotes sense changes in environmental O 2 levels, and the mechanisms involved in controlling these responses, at both the transcriptional and post-transcriptional levels. In this review, we focus on fermentation metabolism in Chlamydomonas and other protists, with only a brief discussion of plant fermentation when relevant, since it is thoroughly discussed in other articles in this volume.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Global changes in mineral transporters in tetraploid switchgrasses ( Panicum virgatum L.)

Switchgrass (Panicum virgatum L) is perennial, C4 grass with great potential as a biofuel crop. An in-depth understanding of the mechanisms that control mineral uptake, distribution and remobilization will benefit sustainable production. Nutrients are mobilized from aerial portions to below-ground crowns and rhizomes as a natural accompaniment to above-ground senescence post seed-set. Mineral uptake and remobilization is dependent on transporters, however, little if any information is available about the specific transporters that are needed and how their relative expression changes over a growing season. Using well-defined classes of mineral transporters, we identified 520 genes belonging to 40 different transporter classes in the tetraploid switchgrass genome. Expression patterns were determined for many of these genes using publically available transcriptomic datasets obtained from both greenhouse and field grown plants. Certain transporters showed strong temporal patterns of expression in distinct developmental stages of the plant. Gene-expression was verified for selected transporters using qRT-PCR. By and large these analyses confirmed the developmental stage-specific expression of these genes. Mineral analyses indicated that K, Fe, Mg, Co, and As had a similar pattern of accumulation with apparent limited remobilization at the end of the growing season. These initial analyses will serve as a foundation for more detailed examination of the nutrient biology of switchgrass.

switchgrass↗
DOE OSTI2014

A dipeptide transporter from the arbuscular mycorrhizal fungus Rhizophagus irregularis is upregulated in the intraradical phase

Arbuscular mycorrhizal fungi (AMF), which form an ancient and widespread mutualistic symbiosis with plants, are a crucial but still enigmatic component of the plant micro biome. Nutrient exchange has probably been at the heart of the success of this plant-fungus interaction since the earliest days of plants on land. To characterize genes from the fungal partner involved in nutrient exchange, and presumably important for the functioning of the AM symbiosis, genome-wide transcriptomic data obtained from the AMF Rhizophagus irregularis were exploited. A gene sequence, showing amino acid sequence and transmembrane domains profile similar to members of the PTR2 family of fungal oligopeptide transporters, was identified and called RiPTR2. The functional properties of RiPTR2 were investigated by means of heterologous expression in Saccharomyces cerevisiae mutants defective in either one or both of its di/tripeptide transporter genes PTR2 and DAL5. These assays showed that RiPTR2 can transport dipeptides such as Ala-Leu, Ala-Tyr or Tyr-Ala. From the gene expression analyses it seems that RiPTR2 responds to different environmental clues when the fungus grows inside the root and in the extraradical phase.

symbiosis↗
DOE OSTI2014

Integration of a constraint-based metabolic model of Brassica napus developing seeds with 13 C-metabolic flux analysis

The use of large-scale or genome-scale metabolic reconstructions for modeling and simulation of plant metabolism and integration of those models with large-scale omics and experimental flux data is becoming increasingly important in plant metabolic research. Here we report an updated version of bna572, a bottom-up reconstruction of oilseed rape (Brassica napus L.; Brassicaceae) developing seeds with emphasis on representation of biomass-component biosynthesis. New features include additional seed-relevant pathways for isoprenoid, sterol, phenylpropanoid, flavonoid, and choline biosynthesis. Being now based on standardized data formats and procedures for model reconstruction, bna572+ is available as a COBRA-compliant Systems Biology Markup Language (SBML) model and conforms to the Minimum Information Requested in the Annotation of Biochemical Models (MIRIAM) standards for annotation of external data resources. Bna572+ contains 966 genes, 671 reactions, and 666 metabolites distributed among 11 subcellular compartments. It is referenced to the Arabidopsis thaliana genome, with gene-protein-reaction (GPR) associations resolving subcellular localization. Detailed mass and charge balancing and confidence scoring were applied to all reactions. Using B. napus seed specific transcriptome data, expression was verified for 78% of bna572+ genes and 97% of reactions. Alongside bna572+ we also present a revised carbon centric model for 13 C-Metabolic Flux Analysis ( 13 C-MFA) with all its reactions being referenced to bna572+ based on linear projections. By integration of flux ratio constraints obtained from 13 C-MFA and by elimination of infinite flux bounds around thermodynamically infeasible loops based on COBRA loopless methods, we demonstrate improvements in predictive power of Flux Variability Analysis (FVA). In conclusion, using this combined approach we characterize the difference in metabolic flux of developing seeds of two B. napus genotypes contrasting in starch and oil content.

13C-metabolic flux analysis↗
DOE OSTI2016

Modulation of medium-chain fatty acid synthesis in Synechococcus sp. PCC 7002 by replacing FabH with a Chaetoceros Ketoacyl-ACP synthase

The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novo assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase Ill increased MCFA synthesis up to fivefold. In conclusion, the level of increase is dependent on promoter strength and culturing conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Photosynthate Regulation of the Root System Architecture Mediated by the Heterotrimeric G Protein Complex in Arabidopsis

Assimilate partitioning to the root system is a desirable developmental trait to control but little is known of the signaling pathway underlying partitioning. A null mutation in the gene encoding the Gβ subunit of the heterotrimeric G protein complex, a nexus for a variety of signaling pathways, confers altered sugar partitioning in roots. While fixed carbon rapidly reached the roots of wild type and agb1-2 mutant seedlings, agb1 roots had more of this fixed carbon in the form of glucose, fructose, and sucrose which manifested as a higher lateral root density. Upon glucose treatment, the agb1-2 mutant had abnormal gene expression in the root tip validated by transcriptome analysis. In addition, PIN2 membrane localization was altered in the agb1-2 mutant. The heterotrimeric G protein complex integrates photosynthesis-derived sugar signaling incorporating both membrane-and transcriptional-based mechanisms. The time constants for these signaling mechanisms are in the same range as photosynthate delivery to the root, raising the possibility that root cells are able to use changes in carbon fixation in real time to adjust growth behavior.

59 BASIC BIOLOGICAL SCIENCES↗