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DOE OSTI2024

Spatial Proteomics towards cellular Resolution

Introduction: Spatial biology is an emerging interdisciplinary field facilitating biological discoveries through the use of spatial omics technologies. Recent advancements in spatial transcriptomics, spatial genomics (e.g. genetic mutations and epigenetic marks), multiplexed immunofluorescence, and spatial metabolomics/lipidomics have enabled high-resolution spatial profiling of gene expression, genetic variation, protein expression, and metabolites/lipids profiles in tissue. These developments contribute to a deeper understanding of the spatial organization within tissue microenvironments at the molecular level. Areas covered: This report provides an overview of the untargeted, bottom-up mass spectrometry (MS)-based spatial proteomics workflow. It highlights recent progress in tissue dissection, sample processing, bioinformatics, and liquid chromatography (LC)-MS technologies that are advancing spatial proteomics toward cellular resolution. Expert opinion: The field of untargeted MS-based spatial proteomics is rapidly evolving and holds great promise. To fully realize the potential of spatial proteomics, it is critical to advance data analysis and develop automated and intelligent tissue dissection at the cellular or subcellular level, along with high-throughput LC-MS analyses of thousands of samples. In conclusion, achieving these goals will necessitate significant advancements in tissue dissection technologies, LC-MS instrumentation, and computational tools.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Single-cell proteomics of Arabidopsis leaf mesophyll reveals dynamic protein responses to water-deficit stress

Background The application of single-cell omics tools to biological systems can provide unique insights into diverse cellular populations and their heterogeneous responses to internal and external perturbations. Thus far, most single-cell studies in plant systems have been limited to RNA-sequencing approaches, which only provide indirect readouts of cellular functions. Results Here, we present a single-cell proteomics workflow for plant cells that integrates tape-sandwich protoplasting, piezoelectric cell sorting, nanoPOTS sample preparation, and ion mobility-based MS data acquisition method for label-free single-cell proteomics analysis of Arabidopsis leaf mesophyll cells. From a single leaf protoplast, over 3,000 proteins were quantified with high precision. The workflow is demonstrated to identify stress associated changes in protein abundance by analyzing 117 protoplasts from well-watered and water-deficit stressed plants. Additionally, we describe a new approach for constructing covarying protein networks at the single-cell level and demonstrate how single-cell protein covariation analysis can reveal previously unrecognized protein functions while also capturing stress-induced changes in protein–protein dynamics. Conclusions The label-free scProteomic approach presented here represents a significant advance through the demonstration of a facile protoplast isolation method combined with deep and precise proteomic coverage of Arabidopsis leaf mesophyll cell types. We believe this study will serve as an informative reference to future plant scProteomic investigations.

Arabidopsis↗

DOE OSTI2026

Multi-omic characterization of a soil microbial consortium reveals critical role of succinate and glutamate metabolism during calcium carbonate precipitation

Microbially induced calcium carbonate precipitation (MICP) holds potential for use in soil stabilization and carbon sequestration, with the overall efficiency of the process being a major determinant for use in many environmental and civil engineering applications. While the biogeochemical pathways and enzymes driving MICP are known, the microbial metabolic networks and community dynamics underlying such precipitation remain poorly characterized. To address this gap, we developed a four-member consortium of soil bacteria (Curtobacterium flaccumfaciens, Rhodococcus qingshengii, Microbacterium sp., and Bacillus toyonensis), termed carbon storing consortium - A (CSC-A), that is capable of MICP. Prior work shows that MICP production is higher in CSC-A compared to the sum of carbonate produced by each member, suggesting carbonate production is driven by consortium dynamics. To that end we used a multi-omic integration approach of genomics, transcriptomics, and metabolomics to investigate potential inter-species interactions that may influence the MICP phenotype. Genomic life history characterizations identified evidence of niche specialization by B. toyonensis and Microbacterium, while metatranscriptomic analysis suggests R. qingshengii is a keystone species during growth in urea. By comparing individual species’ metabolomes to the metabolic profile of a shared well of precipitated metabolites, we identified over 200 metabolites predicted to be produced or consumed by CSC-A members. Integrating both data types to search the KEGG reactome highlighted a network centered around glutamine metabolism and branched chain amino acid biosynthesis under regulation during CSC-A growth in urea. Succinate metabolism was also a major node in this network and laboratory assays confirmed that increasing the amount of succinate in the growth medium leads to increased carbonate precipitation by CSC-A, a critical confirmation of our modeling approach. By isolating and identifying the interconnected metabolic components underlying MICP in CSC-A, we identified keystone taxa, metabolites, and pathways important for future optimization of the application of this consortia to carbonate precipitation.

carbon storing consortium - A (CSC-A)↗

DOE OSTI2019

Metaphenomic Responses of a Native Prairie Soil Microbiome to Moisture Perturbations

Climate change is causing shifts in precipitation patterns in the central grasslands of the United States, with largely unknown consequences on the collective physiological responses of the soil microbial community, i.e., the metaphenome. Here, we used an untargeted omics approach to determine the soil microbial community’s metaphenomic response to soil moisture and to define specific metabolic signatures of the response. Specifically, we aimed to develop the technical approaches and metabolic mapping framework necessary for future systematic ecological studies. We collected soil from three locations at the Konza Long-Term Ecological Research (LTER) field station in Kansas, and the soils were incubated for 15 days under dry or wet conditions and compared to field-moist controls. The microbiome response to wetting or drying was determined by 16S rRNA amplicon sequencing, metatranscriptomics, and metabolomics, and the resulting shifts in taxa, gene expression, and metabolites were assessed. Soil drying resulted in significant shifts in both the composition and function of the soil microbiome. In contrast, there were few changes following wetting. The combined metabolic and metatranscriptomic data were used to generate reaction networks to determine the metaphenomic response to soil moisture transitions. Site location was a strong determinant of the response of the soil microbiome to moisture perturbations. However, some specific metabolic pathways changed consistently across sites, including an increase in pathways and metabolites for production of sugars and other osmolytes as a response to drying. Using this approach, we demonstrate that despite the high complexity of the soil habitat, it is possible to generate insight into the effect of environmental change on the soil microbiome and its physiology and functions, thus laying the groundwork for future, targeted studies. Climate change is predicted to result in increased drought extent and intensity in the highly productive, former tallgrass prairie region of the continental United States. These soils store large reserves of carbon. The decrease in soil moisture due to drought has largely unknown consequences on soil carbon cycling and other key biogeochemical cycles carried out by soil microbiomes. In this study, we found that soil drying had a significant impact on the structure and function of soil microbial communities, including shifts in expression of specific metabolic pathways, such as those leading toward production of osmoprotectant compounds. This study demonstrates the application of an untargeted multi-omics approach to decipher details of the soil microbial community’s metaphenotypic response to environmental perturbations and should be applicable to studies of other complex microbial systems as well.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2025

Metagenome-assembled genomes from soil samples in control and warming plots in Blodgett Forest, CA (2014-2021)

The pathways of carbon transport and loss through and from soils—soil organic matter (SOM) depolymerization to dissolved organic carbon and mineralization to carbon dioxide (CO2)—are fundamentally driven by microbial activity, which is strongly regulated by environmental conditions. As part of LBNL (Lawrence Berkeley National Laboratory) TES (Terrestrial Ecosystem Science) Belowground Biogeochemistry Science Focus Area (SFA), we have established a novel whole-soil long-term warming experiment at the University of California (UC) Blodgett Forest Research Station (Sierra Nevada) in 2014, where we study the role of biogeochemical, microbial and geochemical process interactions in SOM decomposition and stabilization.Here, we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community from soil depth profiles collected from 2014 to 2021 from three paired control and warming plots. We collected soil samples across a range of depth profiles (spanning surface to 90 cm deep) from three paired control and warming plots from a temperate mixed forest in Northern California. Each paired plot had been subjected to experimental warming since June 2014 to simulate a predicted climate change scenario for northern California. 101 soil metagenomes were sequenced at JGI (Joint Genome Institute) and UCSF (University of California San Francisco) Center for Advanced Technology and can be found under the JGI (Joint Genome Institute) GOLD (Genomes Online Database) Sequencing project Gs0151586 and NCBI (National Center for Biotechnology Information) Projects PRJNA1225762 and PRJEB39497. Metagenomes were assembled using JGI (Joint Genome Institute) Metagenome Workflow (10.1128/mSystems.00804-20). For each metagenome, the assembled contigs were binned into genomes using 3 binning algorithms (cocacola, metabat, and maxbin) and the resulting bins were consolidated using dastool. The consolidated bins from all metagenomes were pooled, filtered by completeness (>50%) and contamination (<25%), and dereplicated at 99% ANI (average nucleotide identity) using dRep (https://github.com/MrOlm/drep).The dataset includes a zip file of 2321 MAG (Metagenome Assembled Genome) fasta files, the accession numbers for the underlying metagenomes, and a csv file with MAG (Metagenome Assembled Genome) quality metrics and taxonomic classification (GTDB -Genome Taxonomy Database-RS220). This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type. A sample metadata file (samples.csv) that contains site information has also been included.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2016

Multi‐omics of the Soil Microbiome

TheArctic is characterized by vast amounts of carbon stored in permafrost and isan important focal point for the study of climate change as increasing temperature may accelerate microbially‐mediated release of carbon stored inpermafrost into the atmosphere as CO 2 and CH 4 . Yet surprisingly, very little is known about the vulnerability of permafrost andresponse of microorganisms in the permafrost to their changing environment. To address these knowledge gaps we used omics technologiesto explore permafrost microbial communities, to reveal hitherto unknown soilmicrobial populations and to link them to specific soil biogeochemical processes. For these projects we employed an omics pipeline that includes metagenomics, metatranscriptomics and metaproteomics to determine the phylogenetic and functional gene compositions and their expression in complex, non‐sterile soil samples collected from the field at several locations in Alaska. These studies have presented several challenges due to the unprecedented large size of the datasets and the insufficiency of available bioinformatic tools & databases to analyze, correlate and integrate thedata. To address these challenges we developed a novel functional gene databaseand used different assembly and analysis algorithms to determine key featuresin the datasets. The data reveal the microbial diversity, composition andfunctional potential in permafrost and how the microbial communities respond toclimate change impacts. Support or Funding Information Microbiomes in Transition, LDRD, Pacific Northwest National Laboratories, Richland, Washington

Jansson, Janet K.↗

NASA NTRS2024

Space Algae-2 Ground and Lunar Analog Studies in Preparation for Long-Duration Propagation of Cyanobacteria in Spaceflight

There are numerous applications for microalgae in spaceflight missions and on Earth, such as oxygen production, carbon dioxide removal, nutrition, wastewater processing, and biofuel production. Space Algae-2 aims to test the genetic stability of Arthrospira platensis, commonly known as spirulina, during six-months of continuous culture on the International Space Station. Long-duration exposure to ionizing radiation and microgravity may impact growth, nutrient composition, and genetic stability. The high protein, vitamin, antioxidant content, and radiation resistance make spirulina a promising candidate for bioregenerative life support systems. A concept of operations was developed to grow and harvest algal biomass in space. Preflight testing experiments optimized conditions for an extended growth period in a gas permeable bioreactor bag. Preflight and post-harvest storage methods were developed in addition to a novel cryopreservation method. After sample return, multi-omics analysis will be conducted to determine the mutation rate, gene expression, and the protein and metabolite profile. The concept of operations for Space Algae-2 was tested during a lunar mission simulation within a semi-controlled environment. During a six-day lunar analog mission at the Hawai’i Space Exploration Analog and Simulation (HI-SEAS) A. platensis was successfully grown using flight-like hardware. The cyanobacteria were harvested and used to supplement bread as an example of spirulina biomass utilization. Overall, the data collected from Space Algae-2 will inform potential bioengineering of spirulina for space and terrestrial applications.

Algae↗

NASA NTRSDate not supplied

Space Algae-2 Ground and Lunar Analog Studies in Preparation for Long-Duration Propagation of Cyanobacteria in Spaceflight

Algae↗

DOE OSTI2020

Remodeling of the Histoplasma Capsulatum Membrane Induced by Monoclonal Antibodies

Antibodies play a central role in host immunity by directly inactivating or recognizing an invading pathogen to enhance different immune responses to combat the invader. However, the cellular responses of pathogens to the presence of antibodies are not well-characterized. Here, we used different mass spectrometry techniques to study the cellular responses of the pathogenic fungus Histoplasma capsulatum to monoclonal antibodies (mAb) against HSP60, the surface protein involved in infection. A proteomic analysis of H. capsulatum yeast cells revealed that mAb binding regulates a variety of metabolic and signaling pathways, including fatty acid metabolism, sterol metabolism, MAPK signaling and ubiquitin-mediated proteolysis. The regulation of the fatty acid metabolism was accompanied by increases in the level of polyunsaturated fatty acids, which further augmented the degree of unsaturated lipids in H. capsulatum’s membranes and energy storage lipids, such as triacylglycerols, phosphatidylcholines, phosphatidylethanolamines and phosphatidylinositols. MAb treatment also regulated sterol metabolism by increasing the levels of cholesterol and ergosterol in the cells. We also showed that global changes in the lipid profiles resulted in an increased susceptibility of H. capsulatum to the ergosterol-targeting drug amphotericin B. Overall, our data showed that mAb induction of global changes in the composition of H. capsulatum membranes can potentially impact antifungal treatment during histoplasmosis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Metagenomic analysis reveals global-scale patterns of ocean nutrient limitation

Genomes reveal nutrient stress patterns Within the surface ocean, nitrogen, iron, and phosphorous can all be limiting nutrients for phytoplankton depending on location. Ustick et al. used the prevalence of Prochlorococcus genes involved in nutrient acquisition to develop maps of inferred nutrient stress across the global ocean (see the Perspective by Coleman). They found broad patterns of limitation consistent with an Earth system model and nutrient addition experiments. Leveraging metagenomic data in this manner is an appealing approach that will help to expand our understanding of the biogeochemistry in the vast open ocean. Science , this issue p. 287 ; see also p. 239

Science & Technology - Other Topics↗

DOE OSTI2024

Systematic engineering for production of anti-aging sunscreen compound in Pseudomonas putida

Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO 2 has raised concerns due to their negative effects on human health and the environment. In this study, we aim to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, we methodically selected an appropriate host for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). We enhanced shinorine productivity through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, we improved the shinorine biosynthetic pathway by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, we conducted amino acid feeding experiments under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of 13 C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRSDate not supplied

Space Algae-2 Ground and Lunar Analog Studies in Preparation for Long-Duration Propagation of Cyanobacteria in Spaceflight

There are numerous applications for microalgae in spaceflight missions and on Earth, such as, oxygen production, carbon dioxide removal, nutrition, wastewater processing, and biofuel production. Space Algae-2 aims to test the genetic stability of Arthrospira platensis, commonly known as spirulina, during six-months of continuous culture in spaceflight on the International Space Station. Long-duration exposure to ionizing radiation and microgravity may impact growth, nutrient composition, and genetic stability. The high protein, vitamin, antioxidant content, and radiation resistance make spirulina a promising candidate for bioregenerative life support systems during long-duration missions. A concept of operations was developed to grow and harvest algal biomass in space. Preflight testing experiments were conducted to optimize conditions for an extended growth period in a gas permeable bioreactor bag. Preflight and post-harvest storage methods were developed in addition to a novel cryopreservation method. After sample return, multi-omics analysis will be conducted to determine the mutation rate, gene expression, and protein and metabolite profile. The concept of operations for Space Algae-2 was tested at HI-SEAS (Hawai’i Space Exploration Analog and Simulation) during a six-day lunar analog mission (EMMIHS23, EuroMoonMars, International MoonBase Alliance, HI-SEAS, 2023). A. platensis was grown in the semi-controlled environment using flight-like hardware and solar powered LED lights. Then, the biomass was harvested and used to supplement bread as an example of A. platensis utilization. Overall, the data collected from Space Algae-2 will inform potential bioengineering of spirulina for space and terrestrial applications.

Algae↗

DOE OSTI2025

Metagenome-assembled genomes from Wind River Basin floodplain sediments Riverton, Wyoming site (August 2015)

Microorganisms play a key role in cycling nutrients and contaminants in the terrestrial environment depending on their genetic potential. Here we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community in floodplain sediment samples taken August 29, 2015 at a location (KB1) close to DOE Legacy Management well 855 at the Riverton, Wyoming floodplain site in the Wind River Basin (WRB). The groundwater at this site exhibits persistent U, Mo, and sulfate plumes and is one of the field sites in focus for the SLAC Groundwater Quality SFA program. Sediment samples from a deep soil pit were collected from 0 to 234 cm depth below surface at discrete depths every ~10-20 cm for microbial analyses. 13 metagenomes were sequenced through JGI and can be found under Gold sequencing project: Gs0131241. Metagenomes were assembled, binned, and refined using metawrap to generate MAGs (>50% complete and < 10% contamination based on checkM scores).This dataset includes a zip file of 2216 MAG fasta files and a csv file with quality, taxonomic classification (GTDB RS220), and metagenome accessions for MAGs. This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

Protein N -Glycans in Healthy and Sclerotic Glomeruli in Diabetic Kidney Disease

Diabetes is expected to directly affect renal glycosylation; yet to date, there has not been a comprehensive evaluation of alterations in N-glycan composition in the glomeruli of patients with diabetic kidney disease (DKD). Here, we used untargeted mass spectrometry imaging to identify N-glycan structures in healthy and sclerotic glomeruli in formalin-fixed paraffin-embedded sections from needle biopsies of five patients with DKD and three healthy kidney samples. Regional proteomics was performed on glomeruli from additional biopsies from the same patients to compare the abundances of enzymes involved in glycosylation. Secondary analysis of single-nucleus RNA sequencing (snRNAseq) data were used to inform on transcript levels of glycosylation machinery in different cell types and states. We detected 120 N-glycans, and among them, we identified 12 of these protein post-translated modifications that were significantly increased in glomeruli. All glomeruli-specific N-glycans contained an N-acetyllactosamine epitope. Five N-glycan structures were highly discriminant between sclerotic and healthy glomeruli. Sclerotic glomeruli had an additional set of glycans lacking fucose linked to their core, and they did not show tetra-antennary structures that were common in healthy glomeruli. Orthogonal omics analyses revealed lower protein abundance and lower gene expression involved in synthesizing fucosylated and branched N-glycans in sclerotic podocytes. In snRNAseq and regional proteomics analyses, we observed that genes and/or proteins involved in sialylation and N-acetyllactosamine synthesis were also downregulated in DKD glomeruli, but this alteration remained undetectable by our spatial N-glycomics assay. Integrative spatial glycomics, proteomics, and transcriptomics revealed protein N-glycosylation characteristic of sclerotic glomeruli in DKD.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Inference of cell type-specific gene regulatory networks on cell lineages from single cell omic datasets

Abstract Cell type-specific gene expression patterns are outputs of transcriptional gene regulatory networks (GRNs) that connect transcription factors and signaling proteins to target genes. Single-cell technologies such as single cell RNA-sequencing (scRNA-seq) and single cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq), can examine cell-type specific gene regulation at unprecedented detail. However, current approaches to infer cell type-specific GRNs are limited in their ability to integrate scRNA-seq and scATAC-seq measurements and to model network dynamics on a cell lineage. To address this challenge, we have developed single-cell Multi-Task Network Inference (scMTNI), a multi-task learning framework to infer the GRN for each cell type on a lineage from scRNA-seq and scATAC-seq data. Using simulated and real datasets, we show that scMTNI is a broadly applicable framework for linear and branching lineages that accurately infers GRN dynamics and identifies key regulators of fate transitions for diverse processes such as cellular reprogramming and differentiation.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics

Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with isobaric labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general <100×) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g. 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ~2500 proteins and precise quantification of ~1500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Metagenome-assembled genomes from Wind River Basin floodplain sediments Riverton, Wyoming site (May to September 2017)

Microorganisms play a key role in cycling nutrients and contaminants in the terrestrial environment depending on their genetic potential. Here we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community in floodplain sediment samples taken roughly every month in the period May 18 to September 13 in 2017 at a location (Pit2) close to DOE Legacy Management well 855 at the Riverton, Wyoming floodplain site in the Wind River Basin (WRB). The groundwater at this site exhibits persistent U, Mo, and sulfate plumes and is one of the field sites in focus for the SLAC Groundwater Quality SFA program. Cores were taken with a hand-auger and separated into 5-20 cm segments based on soil horizonation down to 150 cm depth below surface. Each segment was subsampled for microbial analyses. Corresponding 16S rRNA gene amplicon data is available at the NCBI Single Read Archive (SRA) Database BioProject ID PRJNA626616, and soil geochemistry data at doi:10.15485/1631972. 40 metagenomes were sequenced through JGI and can be found under Gold sequencing project: Gs0142591. Metagenomes were assembled, binned, and refined using metawrap to generate MAGs (>50% complete and < 10% contamination based on checkM scores). This dataset includes a zip file of 6993 MAG fasta files and a csv file with quality, taxonomic classification (GTDB RS220), and metagenome accessions for MAGs generated from the Wind River Basin (WRB). This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type.

54 ENVIRONMENTAL SCIENCES↗

NASA NTRS2016

Microarray Data Analysis of Space Grown Arabidopsis Leaves for Genes Important in Vascular Patterning

Venation patterning in leaves is a major determinant of photosynthesis efficiency because of its dependency on vascular transport of photo-assimilates, water, and minerals. Arabidopsis thaliana grown in microgravity show delayed growth and leaf maturation. Gene expression data from the roots, hypocotyl, and leaves of A. thaliana grown during spaceflight vs. ground control analyzed by Affymetrix microarray are available through NASA's GeneLab (GLDS-7). We analyzed the data for differential expression of genes in leaves resulting from the effects of spaceflight on vascular patterning. Two genes were found by preliminary analysis to be up-regulated during spaceflight that may be related to vascular formation. The genes are responsible for coding an ARGOS (Auxin-Regulated Gene Involved in Organ Size)-like protein (potentially affecting cell elongation in the leaves), and an F-box/kelch-repeat protein (possibly contributing to protoxylem specification). Further analysis that will focus on raw data quality assessment and a moderated t-test may further confirm up-regulation of the two genes and/or identify other gene candidates. Plants defective in these genes will then be assessed for phenotype by the mapping and quantification of leaf vascular patterning by NASA's VESsel GENeration (VESGEN) software to model specific vascular differences of plants grown in spaceflight.

venation↗