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At least 199 records · Page 11
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Enhancers in Plant Development, Adaptation and Evolution

Understanding plant responses to developmental and environmental cues is crucial for studying morphological divergence and local adaptation. Gene expression changes, governed by cis-regulatory modules (CRMs) including enhancers, are a major source of plant phenotypic variation. However, while genome-wide approaches have revealed thousands of putative enhancers in mammals, far fewer have been identified and functionally characterized in plants. This review provides an overview of how enhancers function to control gene regulation, methods to predict DNA sequences that may have enhancer activity, methods utilized to functionally validate enhancers and the current knowledge of enhancers in plants, including how they impact plant development, response to environment and evolutionary adaptation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Data for The Stem Cell-Type Transcriptome of Bioenergy Sorghum Reveals the Spatial Regulation of Secondary Cell Wall Networks

Bioenergy sorghum is a low-input, drought-resilient, deep-rooting annual crop that has high biomass yield potential enabling the sustainable production of biofuels, biopower, and bioproducts. Bioenergy sorghum’s 4-5 m stems account for ~80% of the harvested biomass. Stems accumulate high levels of sucrose that could be used to synthesize bioethanol and useful biopolymers if information about stem cell-type gene expression and regulation was available to enable engineering. To obtain this information, Laser Capture Microdissection (LCM) was used to isolate and collect transcriptome profiles from five major cell types that are present in stems of the sweet sorghum Wray. Transcriptome analysis identified genes with cell-type specific and cell-preferred expression patterns that reflect the distinct metabolic, transport, and regulatory functions of each cell type. Analysis of cell-type specific gene regulatory networks (GRNs) revealed that unique TF families contribute to distinct regulatory landscapes, where regulation is organized through various modes and identifiable network motifs. Cell-specific transcriptome data was combined with a stem developmental transcriptome dataset to identify the GRN that differentially activates the secondary cell wall (SCW) formation in stem xylem sclerenchyma and epidermal cells. The cell-type transcriptomic dataset provides a valuable source of information about the function of sorghum stem cell types and GRNs that will enable the engineering of bioenergy sorghum stems.

Software↗
DOE OSTI2023

Cell‐ and development‐specific degradation controls the levels of mixed‐linkage glucan in sorghum leaves

SUMMARY Mixed‐linkage glucan (MLG) is a component of the cell wall (CW) of grasses and is composed of glucose monomers linked by β‐1,3 and β‐1,4 bonds. MLG is believed to have several biological functions, such as the mobilizable storage of carbohydrates and structural support of the CW. The extracellular levels of MLG are largely controlled by rates of synthesis mediated by cellulose synthase‐like (CSL) enzymes, and turnover by lichenases. Economically important crops like sorghum accumulate MLG to variable levels during development. While in sorghum, like other grasses, there is one major MLG synthase (CSLF6), the identity of lichenases is yet unknown. To fill this gap, we identified three sorghum lichenases (SbLCH1‐3) and characterized them in leaves in relation to the expression of SbCSLF6 , and the abundance of MLG and starch. We established that SbLCH1‐3 are secreted to the apoplast, consistent with a role of degrading MLG extracellularly. Furthermore, while SbCSLF6 expression was associated with cell development, the SbLCH genes exhibited distinct development‐, cell‐type‐specific and diel‐regulated expression. Therefore, our study identifies three functional sorghum MLG lichenases and highlights that MLG accumulation in sorghum leaves is likely controlled by the activity of lichenases that tune MLG levels, possibly to suit distinct cell and developmental needs in planta . These findings have important implications for improving the growth, yield, and composition of sorghum as a feedstock.

Plant Sciences↗
DOE OSTI2021

The RIT1 C-terminus associates with lipid bilayers via charge complementarity

RIT1 is a member of the Ras superfamily of small GTPases involved in regulation of cellular signaling. Mutations to RIT1 are involved in cancer and developmental disorders. Like many Ras subfamily members, RIT1 is localized to the plasma membrane. However, RIT1 lacks the C-terminal prenylation that helps many other subfamily members adhere to cellular membranes. For this study, we used molecular dynamics simulations to examine the mechanisms by which the C-terminal peptide (CTP) of RIT1 associates with lipid bilayers. We show that the CTP is unstructured and that its membrane interactions depend on lipid composition. While a 12-residue region of the CTP binds strongly to anionic bilayers containing phosphatidylserine lipids, the CTP termini fray from the membrane allowing for accommodation of the RIT1 globular domain at the membrane-water interface.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A Wox3 -patterning module organizes planar growth in grass leaves and ligules

Grass leaves develop from a ring of primordial initial cells within the periphery of the shoot apical meristem, a pool of organogenic stem cells that generates all of the organs of the plant shoot. At maturity, the grass leaf is a flattened, strap-like organ comprising a proximal supportive sheath surrounding the stem and a distal photosynthetic blade. The sheath and blade are partitioned by a hinge-like auricle and the ligule, a fringe of epidermally derived tissue that grows from the adaxial (top) leaf surface. Together, the ligule and auricle comprise morphological novelties that are specific to grass leaves. Understanding how the planar outgrowth of grass leaves and their adjoining ligules is genetically controlled can yield insight into their evolutionary origins. Here we use single-cell RNA-sequencing analyses to identify a ‘rim’ cell type present at the margins of maize leaf primordia. Cells in the leaf rim have a distinctive identity and share transcriptional signatures with proliferating ligule cells, suggesting that a shared developmental genetic programme patterns both leaves and ligules. Moreover, we show that rim function is regulated by genetically redundant Wuschel-like homeobox3 (WOX3) transcription factors. Higher-order mutations in maize Wox3 genes greatly reduce leaf width and disrupt ligule outgrowth and patterning. Together, these findings illustrate the generalizable use of a rim domain during planar growth of maize leaves and ligules, and suggest a parsimonious model for the homology of the grass ligule as a distal extension of the leaf sheath margin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Evolutionary Analyses of Gene Expression Divergence in Panicum hallii : Exploring Constitutive and Plastic Responses Using Reciprocal Transplants

Abstract The evolution of gene expression is thought to be an important mechanism of local adaptation and ecological speciation. Gene expression divergence occurs through the evolution of cis- polymorphisms and through more widespread effects driven by trans-regulatory factors. Here, we explore expression and sequence divergence in a large sample of Panicum hallii accessions encompassing the species range using a reciprocal transplantation experiment. We observed widespread genotype and transplant site drivers of expression divergence, with a limited number of genes exhibiting genotype-by-site interactions. We used a modified FST–QST outlier approach (QPC analysis) to detect local adaptation. We identified 514 genes with constitutive expression divergence above and beyond the levels expected under neutral processes. However, no plastic expression responses met our multiple testing correction as QPC outliers. Constitutive QPC outlier genes were involved in a number of developmental processes and responses to abiotic environments. Leveraging earlier expression quantitative trait loci results, we found a strong enrichment of expression divergence, including for QPC outliers, in genes previously identified with cis and cis–environment interactions but found no patterns related to trans-factors. Population genetic analyses detected elevated sequence divergence of promoters and coding sequence of constitutive expression outliers but little evidence for positive selection on these proteins. Our results are consistent with a hypothesis of cis-regulatory divergence as a primary driver of expression divergence in P. hallii.

3′ TagSeq↗
DOE OSTI2021

Specialized Plant Growth Chamber Designs to Study Complex Rhizosphere Interactions

The rhizosphere is a dynamic ecosystem shaped by complex interactions between plant roots, soil, microbial communities and other micro- and macro-fauna. Although studied for decades, critical gaps exist in the study of plant roots, the rhizosphere microbiome and the soil system surrounding roots, partly due to the challenges associated with measuring and parsing these spatiotemporal interactions in complex heterogeneous systems such as soil. To overcome the challenges associated with in situ study of rhizosphere interactions, specialized plant growth chamber systems have been developed that mimic the natural growth environment. This review discusses the currently available lab-based systems ranging from widely known rhizotrons to other emerging devices designed to allow continuous monitoring and non-destructive sampling of the rhizosphere ecosystems in real-time throughout the developmental stages of a plant. We categorize them based on the major rhizosphere processes it addresses and identify their unique challenges as well as advantages. We find that while some design elements are shared among different systems (e.g., size exclusion membranes), most of the systems are bespoke and speaks to the intricacies and specialization involved in unraveling the details of rhizosphere processes. We also discuss what we describe as the next generation of growth chamber employing the latest technology as well as the current barriers they face. We conclude with a perspective on the current knowledge gaps in the rhizosphere which can be filled by innovative chamber designs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The cholesterol transporter NPC1 is essential for epigenetic regulation and maturation of oligodendrocyte lineage cells

The intracellular cholesterol transporter NPC1 functions in late endosomes and lysosomes to efflux unesterified cholesterol, and its deficiency causes Niemann–Pick disease Type C, an autosomal recessive lysosomal disorder characterized by progressive neurodegeneration and early death. Here, we use single-nucleus RNA-seq on the forebrain of Npc1 –/– mice at P16 to identify cell types and pathways affected early in pathogenesis. Our analysis uncovers significant transcriptional changes in the oligodendrocyte lineage during developmental myelination, accompanied by diminished maturation of myelinating oligodendrocytes. We identify upregulation of genes associated with neurogenesis and synapse formation in Npc1 –/– oligodendrocyte lineage cells, reflecting diminished gene silencing by H3K27me3. Npc1 –/ – oligodendrocyte progenitor cells reproduce impaired maturation in vitro, and this phenotype is rescued by treatment with GSK-J4, a small molecule inhibitor of H3K27 demethylases. Moreover, mobilizing stored cholesterol in Npc1 –/– mice by a single administration of 2-hydroxypropyl-β-cyclodextrin at P7 rescues myelination, epigenetic marks, and oligodendrocyte gene expression. Our findings highlight an important role for NPC1 in oligodendrocyte lineage maturation and epigenetic regulation, and identify potential targets for therapeutic intervention.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Protein ubiquitination in plant peroxisomes

Protein ubiquitination regulates diverse cellular processes in eukaryotic organisms, from growth and development to stress response. Proteins subjected to ubiquitination can be found in virtually all subcellular locations and organelles, including peroxisomes, single-membrane and highly dynamic organelles ubiquitous in eukaryotes. Peroxisomes contain metabolic functions essential to plants and animals such as lipid catabolism, detoxification of reactive oxygen species (ROS), biosynthesis of vital hormones and cofactors, and photorespiration. Plant peroxisomes possess a complex proteome with functions varying among different tissue types and developmental stages, and during plant response to distinct environmental cues. However, how these diverse functions are regulated at the post-translational level is poorly understood, especially in plants. Here, in this review, we summarized current knowledge of the involvement of protein ubiquitination in peroxisome protein import, remodeling, pexophagy, and metabolism, focusing on plants, and referencing discoveries from other eukaryotic systems when relevant. Based on previous ubiquitinomics studies, we compiled a list of 56 ubiquitinated Arabidopsis peroxisomal proteins whose functions are associated with all the major plant peroxisomal metabolic pathways. This discovery suggests a broad impact of protein ubiquitination on plant peroxisome functions, therefore substantiating the need to investigate this significant regulatory mechanism in peroxisomes at more depths.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Ultraconserved enhancer function does not require perfect sequence conservation

Ultraconserved enhancer sequences show perfect conservation between human and rodent genomes, suggesting that their functions are highly sensitive to mutation. However, current models of enhancer function do not sufficiently explain this extreme evolutionary constraint. We subjected 23 ultraconserved enhancers to different levels of mutagenesis, collectively introducing 1,547 mutations, and examined their activities in transgenic mouse reporter assays. Overall, we find that the regulatory properties of ultraconserved enhancers are robust to mutation. Upon mutagenesis, nearly all (19/23, 83%) still functioned as enhancers at one developmental stage, as did most of those tested again later in development (5/9, 56%). Replacement of endogenous enhancers with mutated alleles in mice corroborated results of transgenic assays, including the functional resilience of ultraconserved enhancers to mutation. Furthermore, our findings show that the currently known activities of ultraconserved enhancers do not necessarily require the perfect conservation observed in evolution and suggest that additional regulatory or other functions contribute to their sequence constraint.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Natural diversity provides a broad spectrum of cyanobacteriochrome-based diguanylate cyclases

Cyanobacteriochromes (CBCRs) are spectrally diverse photosensors from cyanobacteria distantly related to phytochromes that exploit photoisomerization of linear tetrapyrrole (bilin) chromophores to regulate associated signaling output domains. Unlike phytochromes, a single CBCR domain is sufficient for photoperception. CBCR domains that regulate the production or degradation of cyclic nucleotide second messengers are becoming increasingly well characterized. Furthermore, cyclic di-guanosine monophosphate (c-di-GMP) is a widespread small-molecule regulator of bacterial motility, developmental transitions, and biofilm formation whose biosynthesis is regulated by CBCRs coupled to GGDEF (diguanylate cyclase) output domains. In this study, we compare the properties of diverse CBCR-GGDEF proteins with those of synthetic CBCR-GGDEF chimeras. Our investigation shows that natural diversity generates promising candidates for robust, broad spectrum optogenetic applications in live cells. Since light quality is constantly changing during plant development as upper leaves begin to shade lower leaves—affecting elongation growth, initiation of flowering, and responses to pathogens, these studies presage application of CBCR-GGDEF sensors to regulate orthogonal, c-di-GMP-regulated circuits in agronomically important plants for robust mitigation of such deleterious responses under natural growing conditions in the field.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Guided construction of single cell reference for human and mouse lung

Accurate cell type identification is a key and rate-limiting step in single-cell data analysis. Single-cell references with comprehensive cell types, reproducible and functionally validated cell identities, and common nomenclatures are much needed by the research community for automated cell type annotation, data integration, and data sharing. Here, we develop a computational pipeline utilizing the LungMAP CellCards as a dictionary to consolidate single-cell transcriptomic datasets of 104 human lungs and 17 mouse lung samples to construct LungMAP single-cell reference (CellRef) for both normal human and mouse lungs. CellRefs define 48 human and 40 mouse lung cell types catalogued from diverse anatomic locations and developmental time points. We demonstrate the accuracy and stability of LungMAP CellRefs and their utility for automated cell type annotation of both normal and diseased lungs using multiple independent methods and testing data. We develop user-friendly web interfaces for easy access and maximal utilization of the LungMAP CellRefs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Integrating Data From In Vitro New Approach Methodologies for Developmental Neurotoxicity

Abstract In vivo developmental neurotoxicity (DNT) testing is resource intensive and lacks information on cellular processes affected by chemicals. To address this, DNT new approach methodologies (NAMs) are being evaluated, including: the microelectrode array neuronal network formation assay; and high-content imaging to evaluate proliferation, apoptosis, neurite outgrowth, and synaptogenesis. This work addresses 3 hypotheses: (1) a broad screening battery provides a sensitive marker of DNT bioactivity; (2) selective bioactivity (occurring at noncytotoxic concentrations) may indicate functional processes disrupted; and, (3) a subset of endpoints may optimally classify chemicals with in vivo evidence for DNT. The dataset was comprised of 92 chemicals screened in all 57 assay endpoints sourced from publicly available data, including a set of DNT NAM evaluation chemicals with putative positives (53) and negatives (13). The DNT NAM battery provides a sensitive marker of DNT bioactivity, particularly in cytotoxicity and network connectivity parameters. Hierarchical clustering suggested potency (including cytotoxicity) was important for classifying positive chemicals with high sensitivity (93%) but failed to distinguish patterns of disrupted functional processes. In contrast, clustering of selective values revealed informative patterns of differential activity but demonstrated lower sensitivity (74%). The false negatives were associated with several limitations, such as the maximal concentration tested or gaps in the biology captured by the current battery. This work demonstrates that this multi-dimensional assay suite provides a sensitive biomarker for DNT bioactivity, with selective activity providing possible insight into specific functional processes affected by chemical exposure and a basis for further research.

Carstens, Kelly E.↗
DOE OSTI2023

C-terminal conformational changes in SCF-D3/MAX2 ubiquitin ligase are required for KAI2-mediated signaling

Karrikins (KARs) are bioactive molecules derived from burning vegetation. Plants perceive KARs through the hydrolase KARRIKIN INSENSITIVE 2 (KAI2) that interacts with the F-box protein ubiquitin ligase MORE AXILLARY GROWTH 2 (MAX2). MAX2 also plays a role in the perception and signal activation by Strigolactone (SL), a phytohormone controlling various developmental processes in plants. SL also acts as a rhizosphere signal to activate arbuscular mycorrhiza fungi that can be exploited by parasitic plants. kai2 knockouts exhibit distinct developmental defects and therefore KAI2 is hypothesized to perceive an unidentified endogenous ligand provisionally termed KAI2-Ligand (KL). Upon KAR/KL perception, the protein complex of KAI2-MAX2 targets SUPPRESSOR OF MAX2-1/2 (SMAX1)/SMXL2 for proteasomal degradation. Despite the identification of the key components KAI2, MAX2, and SMAX1 in KAR/KL signaling, their mode of interaction and regulation remains elusive. Recently, the regulatory function of the conformational switch of MAX2 C-terminal helix (CTH) in SL signaling has been demonstrated however its role in KAR/KL signaling remained unknown. Here we address the function of MAX2-CTH dynamics both in vitro and in planta and show that the central role of CTH is conserved between SL and KAR/KL signaling pathway.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Convergent Evolution in Breadth of Two V H 6-1-Encoded Influenza Antibody Clonotypes from a Single Donor

Understanding how broadly neutralizing antibodies (bnAbs) to influenza hemagglutinin (HA) naturally develop in humans is critical to the design of universal influenza vaccines. Several classes of bnAbs directed to the conserved HA stem were found in multiple individuals, including one encoded by heavy-chain variable domain V H 6-1. We describe two genetically similar V H 6-1 bnAb clonotypes from the same individual that exhibit different developmental paths toward broad neutralization activity. One clonotype evolved from a germline precursor recognizing influenza group 1 subtypes to gain breadth to group 2 subtypes. The other clonotype recognized group 2 subtypes and developed binding to group 1 subtypes through somatic hypermutation. Crystal structures reveal that the specificity differences are primarily mediated by complementarity-determining region H3 (CDR H3). Thus, while V H 6-1 provides a framework for development of HA stem-directed bnAbs, sequence differences in CDR H3 junctional regions during VDJ recombination can alter reactivity and evolutionary pathways toward increased breadth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Topologically associating domain boundaries are required for normal genome function

Topologically associating domain (TAD) boundaries partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotypes1,2,3, but the overall extent to which this occurs remains unknown. Here we demonstrate that targeted deletions of TAD boundaries cause a range of disruptions to normal in vivo genome function and organismal development. We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11–80 kb in size) from the genome. All deletions examined resulted in detectable molecular or organismal phenotypes, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. We observed changes in local 3D chromatin architecture in 7 of 8 (88%) cases, including the merging of TADs and altered contact frequencies within TADs adjacent to the deleted boundary. For 5 of 8 (63%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and reinforce the critical need to carefully consider the potential pathogenicity of noncoding deletions affecting TAD boundaries in clinical genetics screening.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

RootSlice —A novel functional-structural model for root anatomical phenotypes

Root anatomy is an important determinant of root metabolic costs, soil exploration, and soil resource capture. Root anatomy varies substantially within and among plant species. RootSlice is a multicellular functional-structural model of root anatomy developed to facilitate the analysis and understanding of root anatomical phenotypes. RootSlice can capture phenotypically accurate root anatomy in three dimensions of different root classes and developmental zones, of both monocotyledonous and dicotyledonous species. Several case studies are presented illustrating the capabilities of the model. For maize nodal roots, the model illustrated the role of vacuole expansion in cell elongation; and confirmed the individual and synergistic role of increasing root cortical aerenchyma and reducing the number of cortical cell files in reducing root metabolic costs. Integration of RootSlice for different root zones as the temporal properties of the nodal roots in the whole-plant and soil model OpenSimRoot/maize enabled the multiscale evaluation of root anatomical phenotypes, highlighting the role of aerenchyma formation in enhancing the utility of cortical cell files for improving plant performance over varying soil nitrogen supply. Such integrative in silico approaches present avenues for exploring the fitness landscape of root anatomical phenotypes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Novel nucleocytoplasmic protein O -fucosylation by SPINDLY regulates diverse developmental processes in plants

Here, in metazoans, protein O-fucosylation of Ser/Thr residues was only found in secreted or cell surface proteins, and this post-translational modification is catalyzed by ER-localized protein O-fucosyltransferases (POFUTs) in the GT65 family. Recently, a novel nucleocytoplasmic POFUT, SPINDLY (SPY), was identified in the reference plant Arabidopsis thaliana to modify nuclear transcription regulators DELLAs, revealing a new regulatory mechanism for gene expression. The paralog of AtSPY, SECRET AGENT (SEC), is an O-link-N-acetylglucosamine (GlcNAc) transferase (OGT), which O-GlcNAcylates Ser/Thr residues of target proteins. Both AtSPY and AtSEC are tetratricopeptide repeat-domain-containing glycosyltransferases in the GT41 family. The discovery that AtSPY is a POFUT clarified decades of miss-classification of AtSPY as an OGT. SPY and SEC play pleiotropic roles in plant development, and the interactions between SPY and SEC are complex. SPY-like genes are conserved in diverse organisms, except in fungi and metazoans, suggesting that O-fucosylation is a common mechanism in modulating intracellular protein functions.

59 BASIC BIOLOGICAL SCIENCES↗