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NASA GeneLab: Open Science for Life in Space

The NASA GeneLab project capitalizes on multi-omic technologies to maximize the return on spaceflight experiments. To do this, GeneLab maintains a publicly accessible database (GLDS) that houses spaceflight and spaceflight relevant multi-omics data and collaborates with NASA principal investigators and projects to generate additional omics data. GeneLab houses more than 350 transcriptomic, proteomic, metabolomic and epigenomic datasets from plant, animal and microbial experiments, with a growing number of these having been produced by the GeneLab Sequencing Lab. The GLDS contains rich metadata about each experiment and has integrated radiation dosimetry data from experiments flown on the Space Shuttle, International Space Station, and Free Flying spacecrafts. With the increasing amount and complexity of omics data being generated, GeneLab utilizes community-defined, common models for metadata and terminology so that omics data and results are discoverable and reliably reproducible. GeneLab uses the ISA-Tab specification and semantic model for organizing and representing omics metadata. In addition to metadata standards, data files must be open-source file or common exchange formats to ensure accessibility and usability by all users. To ease data ingestion and transfer, the web-based submission tool allows PIs a user-friendly user interface to curate, organize, and publish their space relevant omics data. In the more recent years, data curation and submission portal has incorporated the FAIR principles making data findable, accessible, interoperable, and reusable. To increase reusability of data, GeneLab has implemented an effort to present processed data in the GLDS in addition to the raw omics data. The processed data will enable interpretation of the data by a larger group of students, scientists and the general public. Standard pipelines for the transformation of raw data into visualizations were developed by four GeneLab Analysis Working Groups (animals, plants, microbes, multi-omics) comprised of over 200 scientists from NASA, industry, and academia. To explore the data, the GLDS provides users various tools for data analysis, collaborative workspace for file storage and sharing, and a visualization portal. The analysis platform built using the Galaxy toolshed provides access to a broad variety of users including those with limited bioinformatics experience and students to learn how to analyze spaceflight omics data. The visualization portal takes GeneLab one step closer to data democratization by removing all bioinformatics requisites to interpret transcriptomics data hosted in the repository. To train the next generation of scientists, NASA offers training programs such as GeneLab 4 High School (GL4HS) and GeneLab 4 Universities. NLM Curation at a Scale Workshop 2022 | NASA GeneLab (GL4U) to teach students bioinformatics and computational biology methods to analyze omics data. Discoveries made using GeneLab have begun and will continue to deepen our understanding of biology, advance the field of genomics, and help to discover cures for diseases, create better diagnostic tools, and ultimately allow astronauts to better withstand the rigors of long-duration spaceflight.

GeneLab↗
DOE OSTI2020

Disentangling Sources of Gene Tree Discordance in Phylogenomic Data Sets: Testing Ancient Hybridizations in Amaranthaceae s.l

Gene tree discordance in large genomic data sets can be caused by evolutionary processes such as incomplete lineage sorting and hybridization, as well as model violation, and errors in data processing, orthology inference, and gene tree estimation. Species tree methods that identify and accommodate all sources of conflict are not available, but a combination of multiple approaches can help tease apart alternative sources of conflict. Here, using a phylotranscriptomic analysis in combination with reference genomes, we test a hypothesis of ancient hybridization events within the plant family Amaranthaceae s.l. that was previously supported by morphological, ecological, and Sanger-based molecular data. The data set included seven genomes and 88 transcriptomes, 17 generated for this study. We examined gene-tree discordance using coalescent-based species trees and network inference, gene tree discordance analyses, site pattern tests of introgression, topology tests, synteny analyses, and simulations. We found that a combination of processes might have generated the high levels of gene tree discordance in the backbone of Amaranthaceae s.l. Furthermore, we found evidence that three consecutive short internal branches produce anomalous trees contributing to the discordance. Overall, our results suggest that Amaranthaceae s.l. might be a product of an ancient and rapid lineage diversification, and remains, and probably will remain, unresolved. This work highlights the potential problems of identifiability associated with the sources of gene tree discordance including, in particular, phylogenetic network methods. Our results also demonstrate the importance of thoroughly testing for multiple sources of conflict in phylogenomic analyses, especially in the context of ancient, rapid radiations. We provide several recommendations for exploring conflicting signals in such situations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Maintaining Genome Integrity during Seed Development in Phaseolus vulgaris L.: Evidence from a Transcriptomic Profiling Study

The maintenance of genome integrity is crucial in seeds, due to the constant challenge of several endogenous and exogenous factors. The knowledge concerning DNA damage response and chromatin remodeling during seed development is still scarce, especially in Phaseolus vulgaris L. A transcriptomic profiling of the expression of genes related to DNA damage response/chromatin remodeling mechanisms was performed in P. vulgaris seeds at four distinct developmental stages, spanning from late embryogenesis to seed desiccation. Of the 14,001 expressed genes identified using massive analysis of cDNA ends, 301 belong to the DNA MapMan category. In late embryogenesis, a high expression of genes related to DNA damage sensing and repair suggests there is a tight control of DNA integrity. At the end of filling and the onset of seed dehydration, the upregulation of genes implicated in sensing of DNA double-strand breaks suggests that genome integrity is challenged. The expression of chromatin remodelers seems to imply a concomitant action of chromatin remodeling with DNA repair machinery, maintaining genome stability. The expression of genes related to nucleotide excision repair and chromatin structure is evidenced during the desiccation stage. An overview of the genes involved in DNA damage response and chromatin remodeling during P. vulgaris seed development is presented, providing insights into the mechanisms used by developing seeds to cope with DNA damage.

seed development↗
DOE OSTI2026

Integrative analysis of CAM photosynthesis reveals its impact on primary metabolism in Yucca

Crassulacean Acid Metabolism (CAM) is an adaptation that temporally separates carbon uptake at night from photosynthesis during the day. CAM has evolved repeatedly across vascular plants, as its emergence may depend on simple regulatory changes to deeply conserved metabolic pathways. Modern CAM research relies heavily on interpretation of transcriptomic data, though regulation occurs at multiple levels following transcription. Additionally, while most research to date has focused on a handful of genes and metabolites in the core CAM pathway, the co-option of conserved regulatory and functional genes is bound to have wide-ranging effects on other aspects of primary metabolism. In this study, we integrate transcriptomic, proteomic, and metabolomic data to compare primary metabolism between the CAM species Yucca aloifolia and closely related C 3 species, Y. filamentosa. We observe minimal correlation between protein abundance and mRNA expression, suggesting significant post-transcriptional regulation in CAM species. We also find evidence of shifts in gene expression and metabolite accumulation outside of the central CAM pathway, suggesting that the shift to CAM has cascading effects across primary metabolism, especially nitrogen metabolism. Our findings provide insights into the metabolic shifts associated with CAM evolution, and highlight the complexity of its regulation at multiple biological levels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Update of the FANTOM web resource: high resolution transcriptome of diverse cell types in mammals

Upon the first publication of the fifth iteration of the Functional Annotation of Mammalian Genomes collaborative project, FANTOM5, we gathered a series of primary data and database systems into the FANTOM web resource (http://fantom.gsc.riken.jp) to facilitate researchers to explore transcriptional regulation and cellular states. In the course of the collaboration, primary data and analysis results have been expanded, and functionalities of the database systems enhanced. We believe that our data and web systems are invaluable resources, and we think the scientific community will benefit for this recent update to deepen their understanding of mammalian cellular organization. We introduce the contents of FANTOM5 here, report recent updates in the web resource and provide future perspectives.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Comparative Phenotyping of Two Commonly Used Chlamydomonas reinhardtii Background Strains: CC-1690 (21gr) and CC-5325 (The CLiP Mutant Library Background)

The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to investigate many essential cellular processes in photosynthetic eukaryotes. Two commonly used background strains of Chlamydomonas are CC-1690 and CC-5325. CC-1690, also called 21gr, has been used for the Chlamydomonas genome project and several transcriptome analyses. CC-5325 is the background strain for the Chlamydomonas Library Project (CLiP). Photosynthetic performance in CC-5325 has not been evaluated in comparison with CC-1690. Additionally, CC-5325 is often considered to be cell-wall deficient, although detailed analysis is missing. The circadian rhythms in CC-5325 are also unclear. To fill these knowledge gaps and facilitate the use of the CLiP mutant library for various screens, we performed phenotypic comparisons between CC-1690 and CC-5325. Our results showed that CC-5325 grew faster heterotrophically in dark and equally well in mixotrophic liquid medium as compared to CC-1690. CC-5325 had lower photosynthetic efficiency and was more heat-sensitive than CC-1690. Furthermore, CC-5325 had an intact cell wall which had comparable integrity to that in CC-1690 but appeared to have reduced thickness. Additionally, CC-5325 could perform phototaxis, but could not maintain a sustained circadian rhythm of phototaxis as CC1690 did. Finally, in comparison to CC-1690, CC-5325 had longer cilia in the medium with acetate but slower swimming speed in the medium without nitrogen and acetate. Our results will be useful for researchers in the Chlamydomonas community to choose suitable background strains for mutant analysis and employ the CLiP mutant library for genome-wide mutant screens under appropriate conditions, especially in the areas of photosynthesis, thermotolerance, cell wall, and circadian rhythms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

A role of GUNs-Involved retrograde signaling in regulating Acetyl-CoA carboxylase 2 in Arabidopsis

Highlights: • The expression level of ACC2 is up-regulated in the mutant of BPG2. • GUN1 and GUN5-mediated plastid signals play a role in the upregulation of ACC2 in the bpg2 mutant. • The level of ACC2 is markedly increased in a GUN-dependent manner under plastid gene expression inhibitor treatment. In Arabidopsis thaliana (Arabidopsis), Acetyl-CoA Carboxylase 2 (ACC2) is a nuclear DNA-encoded and plastid-targeted enzyme that catalyzes the conversion of acetyl-CoA to malonyl-CoA. ACC2 improves plant growth and development when chloroplast translation is impaired. However, little is known about the upstream signals that regulate ACC2. Here, through analyzing the transcriptome changes in brz-insensitive-pale green (bpg) 2-2, a pale-green mutant with impaired chloroplast gene expression resulting from loss of the BPG2 function, we found that the level of ACC2 was significantly up-regulated. Through performing genetic analysis, we further demonstrated that loss of the GENOMES UNCOUPLED 1 (GUN1) or GUN5 function partly perturbed the up-regulation of ACC2 in the bpg2-2 mutant, whereas ABA INSENSITIVE 4 (ABI4)-function-loss had no clear effect on the ACC2 expression. Furthermore, when plants were treated with plastid translation inhibitors, such as lincomycin and spectinomycin, the ACC2 transcriptional level was also markedly increased in a GUN-dependent manner. In conclusion, our results suggested that the GUN-involved plastid-to-nucleus retrograde communication played a role in regulating ACC2 in Arabidopsis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Joint mouse–human phenome-wide association to test gene function and disease risk

Phenome-wide association is a novel reverse genetic strategy to analyze genome-to-phenome relations in human clinical cohorts. Here we test this approach using a large murine population segregating for ~5 million sequence variants, and we compare our results to those extracted from a matched analysis of gene variants in a large human cohort. For the mouse cohort, we amassed a deep and broad open-access phenome consisting of ~4,500 metabolic, physiological, pharmacological and behavioural traits, and more than 90 independent expression quantitative trait locus (QTL), transcriptome, proteome, metagenome and metabolome data sets-by far the largest coherent phenome for any experimental cohort (www.genenetwork.org). Here, we tested downstream effects of subsets of variants and discovered several novel associations, including a missense mutation in fumarate hydratase that controls variation in the mitochondrial unfolded protein response in both mouse and Caenorhabditis elegans , and missense mutations in Col6a5 that underlies variation in bone mineral density in both mouse and human.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Identification of bioactive metabolites using activity metabolomics

The metabolome, the collection of small-molecule chemical entities involved in metabolism, has traditionally been studied with the aim of identifying biomarkers in the diagnosis and prediction of disease. However, the value of metabolome analysis (metabolomics) has been redefined from a simple biomarker identification tool to a technology for the discovery of active drivers of biological processes. It is now clear that the metabolome affects cellular physiology through modulation of other ‘omics’ levels, including the genome, epigenome, transcriptome and proteome. In this Review, we focus on recent progress in using metabolomics to understand how the metabolome influences other omics and, by extension, to reveal the active role of metabolites in physiology and disease. Furthermore, this concept of utilizing metabolomics to perform activity screens to identify biologically active metabolites — which we term activity metabolomics — is already having a broad impact on biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Extraction and biomolecular analysis of dermal interstitial fluid collected with hollow microneedles

Dermal interstitial fluid (ISF) is an underutilized information-rich biofluid potentially useful in health status monitoring applications whose contents remain challenging to characterize. Here, we present a facile microneedle approach for dermal ISF extraction with minimal pain and no blistering for human subjects and rats. Extracted ISF volumes were sufficient for determining transcriptome, and proteome signatures. We noted similar profiles in ISF, serum, and plasma samples, suggesting that ISF can be a proxy for direct blood sampling. Dynamic changes in RNA-seq were recorded in ISF from induced hypoxia conditions. Finally, we report the first isolation and characterization, to our knowledge, of exosomes from dermal ISF. The ISF exosome concentration is 12–13 times more enriched when compared to plasma and serum and represents a previously unexplored biofluid for exosome isolation. This minimally invasive extraction approach can enable mechanistic studies of ISF and demonstrates the potential of ISF for real-time health monitoring applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Biological insights from multi-omics analysis strategies: Complex pleotropic effects associated with autophagy

Research strategies that combine molecular data from multiple levels of genome expression (i.e., multi-omics data), often referred to as a systems biology strategy, has been advocated as a route to discovering gene functions. In this study we conducted an evaluation of this strategy by combining lipidomics, metabolite mass-spectral imaging and transcriptomics data from leaves and roots in response to mutations in two AuTophaGy-related ( ATG ) genes of Arabidopsis . Autophagy is an essential cellular process that degrades and recycles macromolecules and organelles, and this process is blocked in the atg7 and atg9 mutants that were the focus of this study. Specifically, we quantified abundances of ~100 lipids and imaged the cellular locations of ~15 lipid molecular species and the relative abundance of ~26,000 transcripts from leaf and root tissues of WT, atg7 and atg9 mutant plants, grown either in normal (nitrogen-replete) and autophagy-inducing conditions (nitrogen-deficient). The multi-omics data enabled detailed molecular depiction of the effect of each mutation, and a comprehensive physiological model to explain the consequence of these genetic and environmental changes in autophagy is greatly facilitated by the a priori knowledge of the exact biochemical function of the ATG7 and ATG9 proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Systems Level Approaches to Understanding and Manipulating Heterocyst Differentiation in Nostoc Punctiforme: Sites of Hydrogenase and Nitrogenase Synthesis and Activity

Heterocysts are specialized cells that establish a physiologically low oxygen concentration; they function as the sites of oxygen-sensitive nitrogen fixation and hydrogen metabolism in certain filamentous cyanobacteria. They are present at a frequency of less than 10% of the cells and singly in a nonrandom spacing pattern in the filaments. The extent of differential gene expression during heterocyst differentiation was defined by DNA microarray analysis in wild type and mutant cultures of Nostoc punctiforme. The results in wild-type cultures identified two groups of genes; approximately 440 that are unique to heterocyst formation and function, and 500 that respond positively and negatively to the transient stress of nitrogen starvation. Nitrogen fixation is initiated within 24 h after induction, but the cultures require another 24 h before growth is reinitiated. Microarray analyses were conducted on strains with altered expression of three genes that regulate the presence and spacing of heterocysts in the filaments; loss of function or over expression of these genes increases the heterocyst frequency 2 to 3 fold compared to the wild-type. Mutations in the genes hetR and hetF result in the inability to differentiate heterocysts, whereas over expression of each gene individually yields multiple contiguous heterocysts at sites in the filaments; they are positive regulatory elements. Mutation of the gene patN results in an increase in heterocysts frequency, but, in this case, the heterocysts are singly spaced in the filaments with a decrease in the number of vegetative cells in the interval between heterocysts; this is a negative regulatory element. However, over expression of patN resulted in the wild-type heterocyst frequency and spacing pattern. Microarray results indicated HetR and HetF influence the transcription of a common set of about 395 genes, as well as about 350 genes unique to each protein. HetR is known to be a transcriptional regulator and HetF is predicted to be a protease, perhaps operating thorough stability of HetR; thus, the influence of HetF on transcription of a unique set of genes was unanticipated. These two proteins are also found in non-heterocyst-forming filamentous cyanobacteria and the results have implications on their other physiological role(s). The PatN protein is unique to heterocyst-forming cyanobacteria. Cytological analysis indicated PatN is present in only one of the two daughter cells following division, but is present in both cell less than 8 h after division. Microarray analysis indicated only five genes were differentially transcribed in the patN mutant compared to the wild type; three up-regulated genes that are known to influence heterocyst differentiation and two down-regulated genes that have an unassigned function. Mutational analyses indicted the two down-regulated genes do not have a distinct role in heterocyst differentiation. Thus, PatN only indirectly impacts transcription. These databases provide lists of differentially transcribed genes involved in nitrogen starvation and cellular differentiation that can be mined for detailed genetic analysis of the regulation of heterocyst formation and function for subsequent photo-biohydrogen production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

The Populus Superoxide Dismutase Gene Family and Its Responses to Drought Stress in Transgenic Poplar Overexpressing a Pine Cytosolic Glutamine Synthetase (GS1a)

Glutamine synthetase (GS) plays a central role in plant nitrogen assimilation, a process intimately linked to soil water availability. We previously showed that hybrid poplar (Populus tremula X alba, INRA 717-1B4) expressing ectopically a pine cytosolic glutamine synthetase gene (GS1a) display enhanced tolerance to drought. Preliminary transcriptome profiling revealed that during drought, members of the superoxide dismutase (SOD) family were reciprocally regulated in GS poplar when compared with the wild-type control, in all tissues examined. SOD was the only gene family found to exhibit such patterns. In silico analysis of the Populus genome identified 12 SOD genes and two genes encoding copper chaperones for SOD (CCSs). The poplar SODs form three phylogenetic clusters in accordance with their distinct metal co-factor requirements and gene structure. Nearly all poplar SODs and CCSs are present in duplicate derived from whole genome duplication, in sharp contrast to their predominantly single-copy Arabidopsis orthologs. Drought stress triggered plant-wide down-regulation of the plastidic copper SODs (CSDs), with concomitant up-regulation of plastidic iron SODs (FSDs) in GS poplar relative to the wild type; this was confirmed at the activity level. We also found evidence for coordinated downregulation of other copper proteins, including plastidic CCSs and polyphenol oxidases, in GS poplar under drought conditions. Both gene duplication and expression divergence have contributed to the expansion and transcriptional diversity of the Populus SOD/CCS families. Coordinated down-regulation of major copper proteins in drought-tolerant GS poplars supports the copper cofactor economy model where copper supply is preferentially allocated for plastocyanins to sustain photosynthesis during drought. Our results also extend previous findings on the compensatory regulation between chloroplastic CSDs and FSDs, and suggest that this copper-mediated mechanism represents a common response to oxidative stress and other genetic manipulations, as in GS poplars, that affect photosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Improved Biofuel Production through Discovery and Engineering of Terpene Metabolism in Switchgrass

Project Objectives - Of the myriad specialized metabolites that plants deploy to adapt to environmental challenges, terpenes form the largest group. In many major crops, unique terpene blends serve as key stress defenses that directly impact plant fitness and yield. In addition, terpenes, such as bisabolene and pinene, are used for producing renewable biofuels. Essential to advancing a broader use of terpenes for biofuel feedstock engineering is a system-wide knowledge of the diverse biosynthetic machinery and defensive potential of often species-specific terpene blends. The proposed project would merge genome-wide enzyme discovery with comparative –omics, protein structural and plant microbiome studies to define the biosynthesis and stress-defensive functions of the switchgrass (Panicum virgatum) terpene network. These insights would be combined with developing and applying non-transgenic genome editing tools to design plants with desirable terpene blends for higher productivity and biofuel production on marginal lands. As a dedicated lignocellulosic feedstock for U.S. biofuel production with high net energy yield, stress tolerance, and available genome resources, switchgrass is well-suited for devising new avenues for biofuel production. Project Description – The diversity of plant terpene defenses is governed by species-specific families of terpene synthase (TPS) and cytochrome P450 monooxygenase (P450) enzymes. Mining of the switchgrass genome (genotype Alamo) identified ~100 TPS and P450 candidate genes, and combinatorial biochemical analysis of synthesized TPSs and P450s revealed more than a dozen enzymes with common and novel activities. In addition, several identified terpene metabolites and the corresponding transcripts were up-regulated in response to abiotic stressors. These findings demonstrate a unique switchgrass terpene network with probable importance to abiotic stress tolerance, thus providing a large chemical portfolio for optimizing crop resistance, yield, and biofuel composition. Leveraging these preliminary data, we propose to generate a genome-wide map of the switchgrass terpene metabolic network through multi-gene co-expression analyses that allow the efficient cross-validation of TPS and P450 functions. Key enzymes would further be applied to structure-function studies via X-ray protein crystallography, homology modeling and site-directed mutagenesis to gain mechanistic insight into the catalytic specificity of switchgrass terpene metabolism and provide gene and amino acid targets for genome editing. In tandem with terpene pathway discovery, system-wide metabolomics, transcriptomics and proteomics studies in switchgrass accessions of contrasting drought tolerance would define the role of switchgrass terpene metabolism in conferring abiotic stress resilience. Metabolic changes would be assessed in a combined approach of targeted (terpenes) and untargeted metabolite profiling using a high-resolution LC-MS/MS approach, differential gene expression analyses through multiplexed Illumina RNA sequencing, and quantitative analysis of high-priority pathway enzymes using multiple reaction monitoring (MRM). Drawing on these insights, knock-down/out mutants of stress-associated pathway nodes would be generated by optimizing transient virus-induced gene silencing (VIGS) and CRISPR/Cas9 systems under control of the Tobacco Rattle Virus (TRV). The resulting mutant lines would then be analyzed for stress susceptibility and the impact on the root microbiome to define gene functions in planta. Knowledge of terpene pathways, enzyme mechanisms and bioactivities would be applied to enhance switchgrass stress resilience and to tailor-make terpene blends for biofuel production. Here, TRV-enabled CRISPR/Cas9 genome editing, including allele-specific knock-out of redundant genes, engineering of enzyme specificity via structure-guided point mutations, and overexpression of terpene genes relevant to stress-protection or biofuel production, would be used to increase metabolic flux toward desired pathways. Broader Impacts - Integrating the system-wide discovery, mechanistic analysis and non-transgenic genome engineering of the switchgrass terpene network aligns the required steps to unlock the chemical potential of this important metabolite class to generate crops that are more resistant to stress and provide advanced biofuel production in light of rising climate pressures as foreseeable challenges for bioenergy crop cultivation. The proposed project would further offer interdisciplinary student training through active involvement in the project and integration of research concepts and outcomes into newly-developed graduate and undergraduate courses on Plant Biotechnology.

09 BIOMASS FUELS↗
DOE OSTI2020

Insights of Molecular Mechanism of Xylem Development in Five Black Poplar Cultivars

Black poplar (Populus deltoides, P. nigra, and their hybrids) is the main poplar cultivars in China. It offers interesting options of large-scale biomass production for bioenergy due to its rapid growth and high yield. Poplar wood properties were associated with chemical components and physical structures during wood formation. In this study, five poplar cultivars, P. euramericana ‘Zhonglin46’ (Pe1), P. euramericana ‘Guariento’ (Pe2), P. nigra ‘N179’ (Pn1), P. deltoides ‘Danhong’ (Pd1), and P. deltoides ‘Nanyang’ (Pd2), were used to explore the molecular mechanism of xylem development. We analyzed the structural differences of developing xylem in the five cultivars and profiled the transcriptome-wide gene expression patterns through RNA sequencing. The cross sections of the developing xylem showed that the cell wall thickness of developed fiber in Pd1 was thickest and the number of xylem vessels of Pn1 was the least. A total of 10,331 differentially expressed genes were identified among 10 pairwise comparisons of the five cultivars, most of them were related to programmed cell death and secondary cell wall thickening. K-means cluster analysis and Gene Ontology enrichment analysis showed that the genes highly expressed in Pd1 were related to nucleotide decomposition, metabolic process, transferase, and microtubule cytoskeleton; whereas the genes highly expressed in Pn1 were involved in cell wall macromolecule decomposition and polysaccharide binding processes. Based on a weighted gene co-expression network analysis, a large number of candidate regulators for xylem development were identified. And their potential regulatory roles to cell wall biosynthesis genes were validated by a transient overexpression system. This study provides a set of promising candidate regulators for genetic engineering to improve feedstock and enhance biofuel conversion in the bioenergy crop Populus.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Genome expansion by allopolyploidization in the fungal strain Coniochaeta 2T2.1 and its exceptional lignocellulolytic machinery

Abstract Background Particular species of the genus Coniochaeta (Sordariomycetes) exhibit great potential for bioabatement of furanic compounds and have been identified as an underexplored source of novel lignocellulolytic enzymes, especially Coniochaeta ligniaria . However, there is a lack of information about their genomic features and metabolic capabilities. Here, we report the first in-depth genome/transcriptome survey of a Coniochaeta species (strain 2T2.1). Results The genome of Coniochaeta sp. strain 2T2.1 has a size of 74.53 Mbp and contains 24,735 protein-encoding genes. Interestingly, we detected a genome expansion event, resulting ~ 98% of the assembly being duplicated with 91.9% average nucleotide identity between the duplicated regions. The lack of gene loss, as well as the high divergence and strong genome-wide signatures of purifying selection between copies indicates that this is likely a recent duplication, which arose through hybridization between two related Coniochaeta -like species (allopolyploidization). Phylogenomic analysis revealed that 2T2.1 is related Coniochaeta sp. PMI546 and Lecythophora sp. AK0013, which both occur endophytically. Based on carbohydrate-active enzyme (CAZy) annotation, we observed that even after in silico removal of its duplicated content, the 2T2.1 genome contains exceptional lignocellulolytic machinery. Moreover, transcriptomic data reveal the overexpression of proteins affiliated to CAZy families GH11, GH10 (endoxylanases), CE5, CE1 (xylan esterases), GH62, GH51 (α- l -arabinofuranosidases), GH12, GH7 (cellulases), and AA9 (lytic polysaccharide monoxygenases) when the fungus was grown on wheat straw compared with glucose as the sole carbon source. Conclusions We provide data that suggest that a recent hybridization between the genomes of related species may have given rise to Coniochaeta sp. 2T2.1. Moreover, our results reveal that the degradation of arabinoxylan, xyloglucan and cellulose are key metabolic processes in strain 2T2.1 growing on wheat straw. Different genes for key lignocellulolytic enzymes were identified, which can be starting points for production, characterization and/or supplementation of enzyme cocktails used in saccharification of agricultural residues. Our findings represent first steps that enable a better understanding of the reticulate evolution and “eco-enzymology” of lignocellulolytic Coniochaeta species.

09 BIOMASS FUELS↗
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↗