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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Development of high throughput and in vitro assays for analyzing RNA modifications

Modifications on RNAs play major roles in their stability, translation, and enzymatic activity. Despite its importance, the current techniques are insufficient to study the structure and function of RNA modifications. Indeed, the National Academies of Science, Engineering and Medicine indicate that developing new tools and further study the function of RNA modifications is strategically a high priority for advancing science in the coming years (https://www.nationalacademies.org/our-work/toward-sequencing-and-mapping-of-rna-modifications). RNA modifications occur in all domains of life controlling processes such as RNA turnover, translation regulation, cellular defenses and bioproduction. Our preliminary data indicated that the insulin mRNA might get ADP-ribosylated by the ADP-ribosyltransferase PARP12. RNA ADP-ribosylation has been described in Escherichia coli. Combined to the fact that ADP-ribosyltransferase (PARP) genes are conserved throughout evolution we hypothesize that this modification might play essential roles in cells. Therefore, we proposed to develop sequencing techniques and in vitro enzymatic assays to identify and validate ADP-ribosylation motifs and sites. Here we report the development of RNA-seq and qPCR assays to identify ADP-ribosylated RNAs, in addition to a nicotinamide adenosine dinucleotide (NAD – ADP-ribosylation donor) consumption assay and an enzyme-linked immunosorbent assay (ELISA) to measure ADP-ribosyltransferase activity. Testing these assays with the insulin mRNA confirmed that this transcript is ADP-ribosylated. These assays will not only enable studying the function of ADP-ribosylation but can be easily adapted for studying other RNA modifications. This will open opportunities to study RNA modifications in different model systems from bacteria to viruses to plants, bringing insights into their cellular functions and the possibility of targeting them for biotechnological applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Inferring assembly-curving trends of bacterial micro-compartment shell hexamers from crystal structure arrangements

Bacterial microcompartments (BMC) are complex macromolecular assemblies that participate in varied chemical processes in about one fourth of bacterial species. BMC-encapsulated enzymatic activities are segregated from other cell contents by means of semipermeable shells, justifying why BMC are viewed as prototype nano-reactors for biotechnological applications. Herein, we undertook a comparative study of bending propensities of BMC hexamers (BMC-H), the most abundant shell constituents. Published data show that some BMC-H, like β -carboxysomal CcmK, tend to assemble flat whereas other BMC-H often build curved objects. Inspection of available crystal structures presenting BMC-H in tiled arrangements permitted us to identify two major assembly modes with a striking connection with experimental trends. All-atom molecular dynamics (MD) supported that BMC-H bending is triggered robustly only from the arrangement adopted in crystals by BMC-H that experimentally form curved objects, leading to very similar arrangements to those found in structures of recomposed BMC shells. Simulations on triplets of planar-behaving hexamers, which were previously reconfigured to comply with such organization, confirmed that bending propensity is mostly defined by the precise lateral positioning of hexamers, rather than by BMC-H identity. Finally, an interfacial lysine was pinpointed as the most decisive residue in controlling PduA spontaneous curvature. Globally, results presented herein should contribute to improve our understanding of the variable mechanisms of biogenesis characterized for BMC, and of possible strategies to regulate BMC size and shape.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Media Robustness and Scalability of Phosphate Regulated Promoters Useful for Two-Stage Autoinduction in E. coli

A key challenge in synthetic biology is the successful utilization of characterized parts, such as promoters, in different biological contexts. We report the evaluation of the media robustness of a small library of E. coli PhoB regulated promoters that enable heterologous protein production in two-stage cultures. Expression levels were measured both in a rich Autoinduction Broth as well as a minimal mineral salts media. Expression was both media and promoter dependent. Of the 16 promoters tested, 4 were identified to have tightly controlled expression, which was also robust to media formulation. Improved promoter robustness led to more predictable scale up and consistent expression in instrumented bioreactors. Furthermore, this subset of PhoB activated promoters, useful for two-stage autoinduction, highlights the impact of the environment on the performance of biological parts and the importance of robustness testing in synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Reverse β-oxidation pathways for efficient chemical production

Abstract Microbial production of fuels, chemicals, and materials has the potential to reduce greenhouse gas emissions and contribute to a sustainable bioeconomy. While synthetic biology allows readjusting of native metabolic pathways for the synthesis of desired products, often these native pathways do not support maximum efficiency and are affected by complex regulatory mechanisms. A synthetic or engineered pathway that allows modular synthesis of versatile bioproducts with minimal enzyme requirement and regulation while achieving high carbon and energy efficiency could be an alternative solution to address these issues. The reverse β-oxidation (rBOX) pathways enable iterative non-decarboxylative elongation of carbon molecules of varying chain lengths and functional groups with only four core enzymes and no ATP requirement. Here, we describe recent developments in rBOX pathway engineering to produce alcohols and carboxylic acids with diverse functional groups, along with other commercially important molecules such as polyketides. We discuss the application of rBOX beyond the pathway itself by its interfacing with various carbon-utilization pathways and deployment in different organisms, which allows feedstock diversification from sugars to glycerol, carbon dioxide, methane, and other substrates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Xylanolytic metabolism is regulated by coordination of transcription factors XynR and XylR in extremely thermophilic Caldicellulosiruptorales

ABSTRACT Global transcription factors (TFs) control metabolic processes in bacteria to efficiently utilize available carbon. The orderCaldicellulosiruptoraleshas drawn interest due to the ability of its members to degrade components of lignocellulosic biomass. Regulatory reconstruction ofAnaerocellum (f. Caldicellulosiruptor) besciiidentified two major global transcription factors for xylan utilization, XynR and XylR, and the corresponding putative transcription factor binding sites. Recombinant versions of XynR (LacI family) and XylR (ROK family) were subjected to fluorescence polarization (FP) and biolayer interferometry (BLI) analysis to confirm the predicted binding sites. Four XynR sites and two XylR sites were validated, accounting for 20 of 26 genes regulated by XynR and six of seven genes regulated by XylR. Bioinformatic analysis of the individual genes controlled by the two regulators showed an inter-dependent scheme for xylan conversion; the transport of xylooligosaccharides (XOS) is dependent on XylR, while enzymes responsible for hydrolysis are controlled by both regulators. For xylose catabolism by the xylose isomerase-xylulose kinase pathway, regulation is also split, with XylR controlling xylose isomerase and XynR controlling xylokinase. The XynR/XylR regulator pair withinA. besciiis conserved in all sequenced species ofCaldicellulosiruptorales, suggesting similarities in regulating linear xylan conversion. In other xylanolytic thermophiles, XylR homologs control xylan degradation, compared to just 6 out of 26 genes forA. bescii. These results show that two separate regulatory schemes (dual repression) are coordinated byA. besciito effectively regulate the hemicellulose inventory and xylan catabolism. IMPORTANCE To take full advantage of extreme thermophiles as platform metabolic engineering microorganisms, the tools for genetic manipulation must be further developed, and strategies that exploit a better understanding of metabolic regulation need to be discerned.Anaerocellum bescii, the most studied of the extremely thermophilic fermentative anaerobic bacteria that can utilize microcrystalline cellulose, can degrade microcrystalline cellulose and hemicellulose and has been metabolically engineered to convert the resulting sugars to products such as ethanol and acetone. For xylan, in particular, two major global transcription factors (TFs), XynR and XylR, play a role in sugar metabolism, although their predicted regulatory interdependence from bioinformatics analysis has not been elucidated experimentally. Here, fluorescence polarization (FP) and biolayer interferometry (BLI) were used to explore this issue to support metabolic engineering efforts aimed at improving carbohydrate processing to industrial chemicals.

Biotechnology & Applied Microbiology↗
DOE OSTI2024

Nitrous oxide reduction by two partial denitrifying bacteria requires denitrification intermediates that cannot be respired

ABSTRACT Denitrification is a form of anaerobic respiration wherein nitrate (NO 3 − ) is sequentially reduced via nitrite (NO 2 − ), nitric oxide, and nitrous oxide (N 2 O) to dinitrogen gas (N 2 ) by four reductase enzymes. Partial denitrifying bacteria possess only one or some of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here, we tested the denitrifying capabilities of two purple nonsulfur bacteria,Rhodopseudomonas palustrisCGA0092 andRhodobacter capsulatusSB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO 2 − to N 2 , and SB1003 reduced N 2 O to N 2 . For each bacterium, N 2 O reduction could be used both for electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in darkness. However, N 2 O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO 3 − served as a stable, non-catalyzable molecule that was sufficient to activate N 2 O reduction. Using a β-galactosidase reporter, we found that NO 3 − acted, at least in part, by stimulating N 2 O reductase gene expression. In SB1003, NO 2 − but not NO 3 − activated N 2 O reduction, but NO 2 − was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use. IMPORTANCE Denitrification is a form of microbial respiration wherein nitrate is converted via several nitrogen oxide intermediates into harmless dinitrogen gas. Partial denitrifying bacteria, which individually have some but not all denitrifying enzymes, can achieve complete denitrification as a community by cross-feeding nitrogen oxide intermediates. However, the last intermediate, nitrous oxide (N2O), is a potent greenhouse gas that often escapes, motivating efforts to understand and improve the efficiency of denitrification. Here, we found that at least some partial denitrifying N2O reducers can sense and respond to nitrogen oxide intermediates that they cannot otherwise use. The regulatory effects of nitrogen oxides on partial denitrifiers are thus an important consideration in understanding and applying denitrifying bacterial communities to combat greenhouse gas emissions.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

Conformational Dynamics and Catalytic Backups in a Hyper-thermostable Engineered Archaeal Protein Tyrosine Phosphatase

Protein tyrosine phosphatases (PTPs) are a family of enzymes that play important roles in regulating cellular signaling pathways. The activity of these enzymes is regulated by the motion of a catalytic loop that places a critical conserved aspartic acid side chain into the active site for acid–base catalysis upon loop closure. These enzymes also have a conserved phosphate-binding loop that is typically highly rigid and forms a well-defined anion-binding nest. The intimate links between loop dynamics and chemistry in these enzymes make PTPs an excellent model system for understanding the role of loop dynamics in protein function and evolution. In this context, archaeal PTPs, which have often evolved in extremophilic organisms, are highly understudied, despite their unusual biophysical properties. We present here an engineered chimeric PTP (ShufPTP) generated by shuffling the amino acid sequence of five extant hyperthermophilic archaeal PTPs. Despite ShufPTP’s high sequence similarity to its natural counterparts, it presents a suite of unique properties, including high flexibility of the phosphate binding P-loop, facile oxidation of the active-site cysteine, mechanistic promiscuity, and, most notably, hyperthermostability, with a denaturation temperature likely >130 °C (>8 °C higher than the highest recorded growth temperature of any archaeal strain). Our combined structural, biochemical, biophysical, and computational analysis provides insight both into how small steps in evolutionary space can radically modulate the biophysical properties of an enzyme and showcases the tremendous potential of archaeal enzymes for biotechnology, to generate novel enzymes capable of operating under extreme conditions.

archaea↗
DOE OSTI2021

Bioenergy sorghum’s deep roots: A key to sustainable biomass production on annual cropland

Abstract Bioenergy sorghum has high biomass yield potential, drought resilience, good nitrogen use efficiency, and a root system that contributes to the accumulation of soil organic carbon. In this study, field grown bioenergy sorghum root systems were analyzed during the growing season to characterize their depth, biomass, morphology, anatomy, and gene expression profiles. Bioenergy sorghum roots grew continuously during a 155‐day growing season producing ~175 nodal roots, accumulating ~7 Mg of dry biomass per hectare, and reaching >2 m deep in the soil profile. Nodal roots within 20 cm of the stem were 1–5 mm in diameter, whereas roots deeper in soil profiles were enriched in lateral roots with small diameters (~30–500 µm) enabling growth through soil macropores. In older field‐grown plants, roots with intact endodermal, vascular and inner root tissues were surrounded by degraded or aerenchyma‐filled epidermal and cortical cell layers. Transcriptome analysis of nodal, surface, and deep roots identified >2,500 differentially expressed genes involved in root growth, transport, adaptation, defense, and AMF–root interaction. Deep roots (180–240 cm) differentially expressed genes that regulate lateral root growth. Surface roots (0–20 cm) located mid‐row differentially expressed genes involved in nitrate transport, whereas ammonium transport genes were expressed in surface and deep roots and genes involved in phosphate transport were expressed in nodal, surface, and deep roots. Overall, bioenergy sorghum's long growing season enables root systems to grow deeper and accumulate more biomass than annual grain crops such as maize, attributes that could help restore annual cropland soil organic carbon levels and improve soil productivity. Deep roots active in nutrient transport are positioned to take‐up fertilizer leached deep into soil profiles mitigating potential nutrient run‐off. Bioenergy sorghum's large and deep root system is a key to sustainable production of biofuels, biopower, and bioproducts on annual cropland.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

CRISPR/Cas9-based gene activation and base editing in Populus

The genus Populus has long been used for environmental, agroforestry and industrial applications worldwide. Today Populus is also recognized as a desirable crop for biofuel production and a model tree for physiological and ecological research. As such, various modern biotechnologies, including CRISPR/Cas9-based techniques, have been actively applied to Populus for genetic and genomic improvements for traits such as increased growth rate and tailored lignin composition. However, CRISPR/Cas9 has been primarily used as the active Cas9 form to create knockouts in the hybrid poplar clone “717-1B4” (P. tremula x P. alba clone INRA 717-1B4). Alternative CRISPR/Cas9-based technologies, e.g. those involving modified Cas9 for gene activation and base editing, have not been evaluated in most Populus species for their efficacy. Here we employed a deactivated Cas9 (dCas9)-based CRISPR activation (CRISPRa) technique to fine-tune the expression of two target genes, TPX2 and LecRLK-G which play important roles in plant growth and defense response, in hybrid poplar clone “717-1B4” and poplar clone “WV94” (P. deltoides “WV94”), respectively. We observed that CRISPRa resulted in 1.2-fold to 7.0-fold increase in target gene expression through transient expression in protoplasts and Agrobacterium-mediated stable transformation, demonstrating the effectiveness of dCas9-based CRISPRa system in Populus. In addition, we applied Cas9 nickase (nCas9)-based cytosine base editor (CBE) to precisely introduce premature stop codons via C-to-T conversion, with an efficiency of 13%–14%, in the target gene PLATZ which encodes a transcription factor involved in plant fungal pathogen response in hybrid poplar clone “717-1B4”. Overall, we showcase the successful application of CRISPR/Cas-based technologies in gene expression regulation and precise gene engineering in two Populus species, facilitating the adoption of emerging genome editing tools in woody species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A transcriptome software comparison for the analyses of treatments expected to give subtle gene expression responses

Background: In this comparative study we evaluate the performance of four software tools: DNAstar-D (DESeq2), DNAstar-E (edgeR), CLC Genomics and Partek Flow for identification of differentially expressed genes (DEGs) using a transcriptome of E. coli. The RNA-seq data are from the effect of below-background radiation 5.5 nGy total dose (0.2nGy/hr) on E. coli grown shielded from natural radiation 655 m below ground in a pre-World War II steel vault. The gene expression response to three supplemented sources of radiation designed to mimic natural background, 1952 – 5720 nGy in total dose (71–208 nGy/hr), are compared to this “radiation-deprived” treatment. In addition, RNA-seq data of Caenorhabditis elegans nematode from similar radiation treatments was analyzed by three of the software packages. Results: In E. coli, the four software programs identified one of the supplementary sources of radiation (KCl) to evoke about 5 times more transcribed genes than the minus-radiation treatment (69–114 differentially expressed genes, DEGs), and so the rest of the analyses used this KCl vs “Minus” comparison. After imposing a 30-read minimum cutoff, one of the DNAStar options shared two of the three steps (mapping, normalization, and statistic) with Partek Flow (they both used median of ratios to normalize and the DESeq2 statistical package), and these two programs identified the highest number of DEGs in common with each other (53). In contrast, when the programs used different approaches in each of the three steps, between 31 and 40 DEGs were found in common. Regarding the extent of expression differences, three of the four programs gave high fold-change results (15–178 fold), but one (DNAstar’s DESeq2) resulted in more conservative fold-changes (1.5–3.5). In a parallel study comparing three qPCR commercial validation software programs, these programs also gave variable results as to which genes were significantly regulated. Similarly, the C. elegans analysis showed exaggerated fold-changes in CLC and DNAstar’s edgeR while DNAstar-D was more conservative. Conclusions: Regarding the extent of expression (fold-change), and considering the subtlety of the very low level radiation treatments, in E. coli three of the four programs gave what we consider exaggerated fold-change results (15 – 178 fold), but one (DNAstar’s DESeq2) gave more realistic fold-changes (1.5–3.5). When RT-qPCR validation comparisons to transcriptome results were carried out, they supported the more conservative DNAstar-D’s expression results. When another model organism’s (nematode) response to these radiation differences was similarly analyzed, DNAstar-D also resulted in the most conservative expression patterns. Therefore, we would propose DESeq2 (“DNAstar-D”) as an appropriate software tool for differential gene expression studies for treatments expected to give subtle transcriptome responses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Engineering quantitative stomatal trait variation and local adaptation potential by cis‐regulatory editing

Summary Cis‐regulatory element editing can generate quantitative trait variation that mitigates extreme phenotypes and harmful pleiotropy associated with coding sequence mutations. Here, we applied a multiplexed CRISPR/Cas9 approach, informed by bioinformatic datasets, to generate genotypic variation in the promoter ofOsSTOMAGEN, a positive regulator of rice stomatal density. Engineered genotypic variation corresponded to broad and continuous variation in stomatal density, ranging from 70% to 120% of wild‐type stomatal density. This panel of stomatal variants was leveraged in physiological assays to establish discrete relationships between stomatal morphological variation and stomatal conductance, carbon assimilation and intrinsic water use efficiency in steady‐state and fluctuating light conditions. Additionally, promoter alleles were subjected to vegetative drought regimes to assay the effects of the edited alleles on developmental response to drought. Notably, the capacity for drought‐responsive stomatal density reprogramming instomagenand two cis‐regulatory edited alleles was reduced. Collectively our data demonstrate that cis‐regulatory element editing can generate near‐isogenic trait variation that can be leveraged for establishing relationships between anatomy and physiology, providing a basis for optimizing traits across diverse environments.

Biotechnology & Applied Microbiology↗
DOE OSTI2023

Tripogon loliiformis tolerates rapid desiccation after metabolic and transcriptional priming during initial drying

Abstract Crop plants and undomesticated resilient species employ different strategies to regulate their energy resources and growth. Most crop species are sensitive to stress and prioritise rapid growth to maximise yield or biomass production. In contrast, resilient plants grow slowly, are small, and allocate their resources for survival in challenging environments. One small group of plants, termed resurrection plants, survive desiccation of their vegetative tissue and regain full metabolic activity upon watering. However, the precise molecular mechanisms underlying this extreme tolerance remain unknown. In this study, we employed a transcriptomics and metabolomics approach, to investigate the mechanisms of desiccation tolerance in Tripogon loliiformis , a modified desiccation-tolerant plant, that survives gradual but not rapid drying. We show that T. loliiformis can survive rapid desiccation if it is gradually dried to 60% relative water content (RWC). Furthermore, the gene expression data showed that T. loliiformis is genetically predisposed for desiccation in the hydrated state, as evidenced by the accumulation of MYB, NAC, bZIP, WRKY transcription factors along with the phytohormones, abscisic acid, salicylic acid, amino acids (e.g., proline) and TCA cycle sugars during initial drying. Through network analysis of co-expressed genes, we observed differential responses to desiccation between T. loliiformis shoots and roots. Dehydrating shoots displayed global transcriptional changes across broad functional categories, although no enrichment was observed during drying. In contrast, dehydrating roots showed distinct network changes with the most significant differences occurring at 40% RWC. The cumulative effects of the early stress responses may indicate the minimum requirements of desiccation tolerance and enable T. loliiformis to survive rapid drying. These findings potentially hold promise for identifying biotechnological solutions aimed at developing drought-tolerant crops without growth and yield penalties.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Herbaspirillum rubrisubalbicans as a Phytopathogenic Model to Study the Immune System of Sorghum bicolor

Herbaspirillum rubrisubalbicans is the causal agent of red stripe disease (RSD) and mottle stripe disease of sorghum and sugarcane, respectively. In all, 63 genotypes of Sorghum bicolor were inoculated with H. rubrisubalbicans, with 59 showing RSD symptoms. Quantitative trait loci (QTL) analysis in a recombinant inbred line (RIL) population identified several QTL associated with variation in resistance to RSD. RNA sequencing analysis identified a number of genes whose transcript levels were differentially regulated during H. rubrisubalbicans infection. Among those genes that responded to H. rubrisubalbicans inoculation were many involved in plant–pathogen interactions such as leucine-rich repeat receptors, mitogen-activated protein kinase 1, calcium-binding proteins, transcriptional factors (ethylene-responsive element binding factor), and callose synthase. Pretreatment of sorghum leaves with the pathogen-associated molecular pattern (PAMP) molecules flg22 and chitooctaose provided protection against subsequent challenge with the pathogen, suggesting that PAMP-triggered immunity plays an important role in the sorghum immunity response. These data present baseline information for the use of the genetically tractable H. rubrisubalbicans–sorghum pathosystem for the study of innate immunity and disease resistance in this important grain and bioenergy crop. Information gained from the use of this system is likely to be informative for other monocots, including those more intractable for experimental study (e.g., sugarcane).

Biochemistry & Molecular Biology↗
DOE OSTI2020

Combining Random Mutagenesis and Metabolic Engineering for Enhanced Tryptophan Production in Synechocystis sp. Strain PCC 6803

Tryptophan (Trp) is an essential aromatic amino acid that has value as an animal feed supplement, as the amount found in plant-based sources is insufficient. An alternative to production by engineered microbial fermentation is to have tryptophan biosynthesized by a photosynthetic microorganism that could replace or supplement both the plant and industrially used microbes. We selected Synechocystis sp. strain PCC 6803, a model cyanobacterium, as the host and studied metabolic engineering and random mutagenesis approaches. Previous work on engineering heterotrophic microbes for improved Trp titers has targeted allosteric feedback regulation in enzymes 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS) and anthranilate synthase (AS) as major bottlenecks in the shikimate pathway. In this work, the genes encoding feedback-resistant enzymes from Escherichia coli, aroG fbr and trpE fbr , were overexpressed in the host wild-type (WT) strain. Separately, the WT strain was subjected to random mutagenesis and selection using an amino acid analog to isolate tryptophan-overproducing strains. The randomly mutagenized strains were sequenced in order to identify the mutations that resulted in the desirable phenotypes. Interestingly, the tryptophan overproducers had mutations in the gene encoding chorismate mutase (CM), which catalyzes the conversion of chorismate to prephenate. The best tryptophan overproducer from random mutagenesis was selected as a host for metabolic engineering where aroG fbr and trpE fbr were overexpressed. The best strain developed produced 212 ± 23 mg/liter of tryptophan after 10 days of photoautotrophic growth under 3% (vol/vol) CO2. We demonstrated that a combination of random mutagenesis and metabolic engineering was superior to either individual approach.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Comparing four heat-inducible promoters in stably transformed sugarcane regarding spatial and temporal control of transgene expression reveals candidates to drive stem-preferred transgene expression

Small heat shock protein (sHSP) promoters contain cis-regulatory elements that facilitate transcription in response to heat stress, making them valuable tools for functional studies through controlled gene expression and the precise regulation of gene-editing tools or morphogenic regulators. To evaluate their utility, GUS reporter gene expression driven by four plant-sourced HSP promoters (p GmHSP17.5 , p HvHSP17 , p ZmHSP17.7 , and p ZmHSP26 ) was compared across various tissues of stably transformed sugarcane before and after heat treatment. At 22°C, all promoters showed minimal activity in leaves and roots, although p ZmHSP17.7 and p HvHSP17 displayed moderate expression in stems. Following heat treatment, all promoters exhibited their highest activity in stems, followed by leaves and roots. In stem tissues, p GmHSP17.5 displayed heat-induced uidA expression comparable to the constitutive p ZmUbi promoter. Notably, heat-induced reporter gene activity in stem middle sections of single-copy transgenic lines containing p ZmHSP17.7 , p HvHSP17 , or p ZmHSP26 exceeded p ZmUbi -derived uidA activity by 9.7-fold, 3.8-fold, and 3.0-fold, respectively, with 346- to 3,672-fold induction compared to control conditions. Most promoters showed peak expression in the middle sections of the stem, while p HvHSP17 was the most active in the stem apices. Histochemical analysis revealed that p ZmHSP17.7 and p HvHSP17 were active in both parenchyma cells and vascular bundles within sugarcane stems. Among leaf tissues, mature leaves exhibited greater expression than senescing or immature leaves, while root activity remained consistently minimal across all promoters. Temperature-course experiments identified distinct activation thresholds: 34°C–36°C for p ZmHSP17.7 , 36°C for p ZmHSP26 , 36°C–38°C for p HvHSP17 , and 40°C–42°C for p GmHSP17.5 . Drought stress also induced reporter gene transcription in stems under HSP promoters, although with lower fold induction than heat treatment. These findings provide valuable tools for gene function studies and biotechnology applications, including heat stress tolerance research, controlled transgene expression in metabolic engineering, precision gene editing, and developmental biology studies.

60 APPLIED LIFE 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 OSTI2022

A synthetic cell density signal can drive proliferation in chick embryonic tendon cells and tendon cells from a full size rooster can produce high levels of procollagen in cell culture

Cell density signaling drives tendon morphogenesis by regulating both procollagen production and cell proliferation. The signal is composed of a small, highly conserved protein (SNZR P) tightly bound to a tissue-specific, unique lipid (SNZR L). This allows the complex (SNZR PL) to bind to the membrane of the cell and locally diffuse over a radius of ~1 mm. The cell produces low levels of this signal but the binding to the membrane increases with the number of tendon cells in the local environment. In this article SNZR P was produced in E.coli and SNZR L was chemically synthesized. The two bind together when heated to 60 °C in the presence of Ca ++ and Mg ++ and the synthesized SNZR PL at ng/ml levels can replace serum. Adding SNZR PL to the medium was also tested on primary tendon cells from adult roosters. The older cells were in a maintenance state in vivo and in cell culture they proliferate more slowly than embryonic cells. Nevertheless, after reaching a moderately high cell density, they produced high levels of procollagen similar to the embryonic cells. This data was not expected from older cells but suggests that adult tendon cells can regenerate the tissue after injury when given the correct signals.

59 BASIC BIOLOGICAL SCIENCES↗