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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

PPT1-mediated plastidic phospho enol pyruvate import enhances fatty acid biosynthesis in sugar-rich tissues

Metabolic engineering of vegetative tissues for lipid production holds transformative potential for sustainable biofuels, yet achieving sufficient yields remains challenging. Here, we present a strategy to enhance fatty acid synthesis by redirecting cytosolic phospho enol pyruvate (PEP) into plastids through overexpression of the plastidial phospho enol pyruvate/phosphate translocator ( PPT1 ) in vegetative tissues of Arabidopsis thaliana . Integrated metabolomic and transcriptomic analyses revealed that AtPPT1 overexpression alleviated metabolite overaccumulation in high-sugar tissues, consistent with enhanced carbon flux coordination between the cytosol and chloroplast. Notably, phosphofructokinase activity, a key step in glycolysis, was elevated, linking plastidic PEP import to increased glycolytic throughput. In Arabidopsis, overexpression of AtPPT1 increased fatty acid content and lipid droplet accumulation in the sugar-accumulating sweet11;12;13 mutant, but not in wild-type Col-0. Together, these findings establish PEP redirection as an effective strategy to boost fatty acid and lipid production in sugar-rich vegetative tissues and provide a complementary metabolic module for future lipid-engineering efforts.

Arabidopsis thaliana↗
DOE OSTI2024

An alternate route for cellulose microfibril biosynthesis in plants

Similar to cellulose synthases (CESAs), cellulose synthase–like D (CSLD) proteins synthesize β-1,4-glucan in plants. CSLDs are important for tip growth and cytokinesis, but it was unknown whether they form membrane complexes in vivo or produce microfibrillar cellulose. We produced viable CESA-deficient mutants of the moss Physcomitrium patens to investigate CSLD function without interfering CESA activity. Microscopy and spectroscopy showed that CESA-deficient mutants synthesize cellulose microfibrils that are indistinguishable from those in vascular plants. Correspondingly, freeze-fracture electron microscopy revealed rosette-shaped particle assemblies in the plasma membrane that are indistinguishable from CESA-containing rosette cellulose synthesis complexes (CSCs). Our data show that proteins other than CESAs, most likely CSLDs, produce cellulose microfibrils in P. patens protonemal filaments. The data suggest that the specialized roles of CSLDs in cytokinesis and tip growth are based on differential expression and different interactions with microtubules and possibly Ca 2+ , rather than structural differences in the microfibrils they produce.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Creating multifunctional synthetic lichen platforms for sustainable biosynthesis of biofuel precursors

In this project, we were creating a sustainable platform for biofuel production, utilizing carbon-fixing autotrophs to supply oxygen and organic substrates to heterotrophic partners, which in turn produce carbon dioxide to feed the autotrophs. This symbiotic lichen community could lower the input cost, optimize metabolic exchanges and improve the generation of biofuel precursors through multi-omics driven genetic engineering. The cyanobacteria Synechococcus elongatus (S. elongatus) was used as the primary autotroph to provide oxygen and organic substrates, especially sucrose, to a co-culture system. The strain with overexpression of sucrose transporter cscB demonstrated a significant increase in sucrose production under salt stress as what we expected. We also implemented 13C metabolic flux analysis on the sucrose secreting strain S. elongatus cscB-NaCl. Next, transporters proteins like glutamate exporter mscCG from Corynebacterium glutamicum was overexpressed in S. elongatus to improve metabolite exchange.

Betenbaugh, Michael↗
DOE OSTI2025

Understanding and Harnessing the Robustness of Undomesticated Yarrowia lipolytica Strains for Biosynthesis of Designer Bioesters (Final Report)

This project seeks to elucidate and harness the exceptional robustness of novel and undomesticated Y. lipolytica isolates, which were identified from a genetic diversity screening for compatibility with bioenergy development. Bioenergy-relevant isolates were further developed as microbial platforms for efficient conversion of undetoxified biomass hydrolysates into designer bio-esters continuously recovered by solvent extraction. The project has three major goals. Goal 1. Elucidate and enhance the endogenous metabolism of Y. lipolytica for superior growth, sugar utilization, and lipid accumulation in undetoxified biomass hydrolysates under hypoxic conditions. Goal 2. Understand and enhance the underlying mechanism of exceptional tolerance of Y. lipolytica to organic solvents. Goal 3. Elucidate and rewire endogenous metabolism of the most robust Y. lipolytica strains for effective conversion of accumulating lipids to designer bio-esters. Significant progress has been made toward completing all research goals. We elucidated and optimized the robustness of Y. lipolytica by utilizing mixed C5 and C6 sugars in switchgrass hydrolysates (SGH) for lipid production (Aim 1). We conducted extensive omics analysis to investigate how genetic diversity among Yarrowia strains, derived from natural isolates or developed through adaptive laboratory evolution, influences lipid production when utilizing SGH. In Aim 2, novel mechanisms and underlying genetics were discovered that enabled Yarrowia strains to thrive in cultures containing high ionic liquid (IL) concentrations. Amongst other novel findings, it was found that sterols strengthened cell membranes to confer IL toxicity resistance, specifically via increased ergosterol content upon exposure to IL. In Aim 3, mechanistic studies elucidated how Y. lipolytica utilized intracellular lipids and alkanes/alkenes, leading to our discovery of novel enzymes and pathways for making short-chain esters. Most notably, thermostable chloramphenicol transferases were repurposed to function as alcohol acetyltransferases in Y. lipolytica, as well as the Gram-negative and Gram-positive bacteria Escherichia coli and thermophile Clostridium thermocellum, respectively.

09 BIOMASS FUELS↗
DOE OSTI2025

Seed coat transcriptomic profiling of 5-593, a genotype important for genetic studies of seed coat color and patterning in common bean ( Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) market classes have distinct seed coat colors, which are directly related to the diverse flavonoids found in the mature seed coat. To understand and elucidate the molecular mechanisms underlying the regulation of seed coat color, RNA-Seq data was collected from the black bean 5-593 and used for a differential gene expression and enrichment analysis from four different seed coat color development stages. 5-593 carries dominant alleles for 10 of the 11 major genes that control seed coat color and expression and has historically been used to develop introgression lines used for seed coat genetic analysis. Pairwise comparison among the four stages identified 6,294 differentially expressed genes (DEGs) varying from 508 to 5,780 DEGs depending on the compared stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that phenylpropanoid biosynthesis, flavonoid biosynthesis, and plant hormone signal transduction comprised the principal pathways expressed during bean seed coat pigment development. Transcriptome analysis suggested that most structural genes for flavonoid biosynthesis and some potential regulatory genes were significantly differentially expressed. Further studies detected 29 DEGs as important candidate genes governing the key enzymatic flavonoid biosynthetic pathways for common bean seed coat color development. Additionally, four gene models, Pv5-593.02G016100, 593.02G078700, Pv5-593.02G090900, and Pv5-593.06G121300, encode MYB-like transcription factor family protein were identified as strong candidate regulatory genes in anthocyanin biosynthesis which could regulate the expression levels of some important structural genes in flavonoid biosynthesis pathway. These findings provide a framework to draw new insights into the molecular networks underlying common bean seed coat pigment development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Polyyne production is regulated by the transcriptional regulators PgnC and GacA in Pseudomonas protegens Pf-5

ABSTRACT Polyynes produced by bacteria have promising applications in agriculture and medicine due to their potent antimicrobial activities. Polyyne biosynthetic genes have been identified inPseudomonasandBurkholderia. However, the molecular mechanisms underlying the regulation of polyyne biosynthesis remain largely unknown. In this study, we used a soil bacteriumPseudomonas protegensPf-5, which was recently reported to produce polyyne called protegenin, as a model to investigate the regulation of bacterial polyyne production. Our results show that Pf-5 controls polyyne production at both the pathway-specific level and a higher global level. Mutation ofpgnC, a transcriptional regulatory gene located in the polyyne biosynthetic gene cluster, abolished polyyne production. Gene expression analysis revealed that PgnC directly activates the promoter of polyyne biosynthetic genes. The production of polyyne also requires a global regulator GacA. Mutation ofgacAdecreased the translation of PgnC, which is consistent with the result thatpgnCleader mRNA bound directly to RsmE, an RNA-binding protein negatively regulated by GacA. These results suggest that GacA induces the expression of the PgnC regulator, which in turn activates polyyne biosynthesis. Additionally, the polyyne-producing strain of Pf-5, but not the polyyne-nonproducing strain, could inhibit a broad spectrum of bacteria including both Gram-negative and Gram-positive bacteria. IMPORTANCE Antimicrobial metabolites produced by bacteria are widely used in agriculture and medicine to control plant, animal, and human pathogens. Although bacteria-derived polyynes have been identified as potent antimicrobials for decades, the molecular mechanisms by which bacteria regulate polyyne biosynthesis remain understudied. In this study, we found that polyyne biosynthesis is directly activated by a pathway-specific regulator PgnC, which is induced by a global regulator GacA through the RNA-binding protein RsmE inPseudomonas protegens. To our knowledge, this work is the first comprehensive study of the regulatory mechanisms of bacterial polyyne biosynthesis at both pathway-specific level and global level. The discovered molecular mechanisms can help us optimize polyyne production for agricultural or medical applications.

Biotechnology & Applied Microbiology↗
DOE OSTI2025

Complete replacement of Arabidopsis oil-producing enzymes with heterologous diacylglycerol acyltransferases

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1) share responsibility for triacylglycerol (TAG) biosynthesis, and their selectivities control TAG fatty acid (FA) compositions. For rational metabolic engineering of seed oils, replacing endogenous TAG biosynthesis with exogenous enzymes containing different substrate FA selectivities is desirable; however, the dgat1-1/pdat1-2 double mutant is pollen lethal. Here, we evaluated the ability of 3 DGAT1s, from phylogenetically diverse plants with distinct TAG assembly processes, to completely replace endogenous TAG biosynthesis in Arabidopsis ( Arabidopsis thaliana ). We transformed dgat1-1 mutant plants with expression constructs for DGAT1 s from Camelina sativa , Physaria fendleri , and castor ( Ricinus communis ). Transgene expression was properly “contextualized” by using a previously determined minimum necessary expression unit containing the promoter/5′ UTR and first intron of native AtDGAT1 ; both of these DNA elements are essential for pollen expression. Next, we crossed homozygous lines with a DGAT1/DGAT1/PDAT1/pdat1-2 parent. C. sativa and P. fendleri DGAT1s restored the FA compositions and transcriptional differences of dgat1-1 to near wild-type and rescued the dgat1-1/pdat1-2 pollen lethality. R. communis DGAT1 was active in dgat1-1 seeds but produced unique oil profiles and alterations in the expression of lipid metabolic genes; it also failed to rescue dgat1-1/pdat1-2 lethality. This study confirms that the promoter and first intron of AtDGAT1 can modulate the expression of foreign DGAT1 genes to fit the correct spatiotemporal profile necessary for completely replacing endogenous TAG biosynthesis. Furthermore, it demonstrates an additional layer of unexpected enzyme incompatibility between oilseed lineages, which may complicate bioengineering approaches that seek to replace essential genes with orthologs.

McGuire, Sean T. [Washington State Univ., Pullman,↗
DOE OSTI2026

Nitrogen Status Rewires Transcriptional Regulation of Dhurrin, a Dual‐Purpose Defense Metabolite in Sorghum bicolor

Dhurrin, a cyanogenic glucoside, plays an important role in Sorghum bicolor physiology and defense. The concentration of dhurrin in sorghum is influenced by both nitrogen status and stage of plant organ development. While nitrogen resupply activates the expression of genes for dhurrin biosynthesis, the molecular mechanisms underlying this regulation remain unclear. In this study, we investigated the transcriptional response of sorghum to nitrogen resupply following growth under nitrogen-limiting conditions. Using a time-course design, we measured hydrogen cyanide potential (HCNp), growth, and nitrate content at 0-, 2-, 6-, 12-, 24-, 36-, 48-, and 60-h after resupply and collected tissue for RNAseq analysis in parallel for analysis of gene expression and construction of gene regulatory networks (GRNs). HCNp (mg g −1 DW) increased significantly in leaf and stem tissues following nitrogen resupply, with increases in the leaf partially driven by continued declines in controls under ongoing nitrogen stress. Expression of the dhurrin pathway genes was upregulated in leaves from 24 h after nitrogen resupply, with diel expression patterns observable over the remaining time points. No upregulation was observed in roots or stems, suggesting that developmental context overrides environmental cues. GRN analysis identified candidate transcription factors regulating dhurrin biosynthesis genes, including members of the MYB, bZIP, and GARP-type transcription factor families. Some of these candidate transcription factors may be involved in relieving senescence-associated suppression of dhurrin biosynthesis and link nitrogen signaling to pathway activation. These findings provide new insight into the nitrogen-responsive regulation of dhurrin in sorghum, highlighting candidate regulators for future functional characterization.

S. bicolor↗
DOE OSTI2024

Chlamydomonas cells transition through distinct Fe nutrition stages within 48 h of transfer to Fe-free medium

Low iron (Fe) bioavailability can limit the biosynthesis of Fe-containing proteins, which are especially abundant in photosynthetic organisms, thus negatively affecting global primary productivity. Understanding cellular coping mechanisms under Fe limitation is therefore of great interest. For this paper, we surveyed the temporal responses of Chlamydomonas ( Chlamydomonas reinhardtii ) cells transitioning from an Fe-rich to an Fe-free medium to document their short and long-term adjustments. While slower growth, chlorosis and lower photosynthetic parameters are evident only after one or more days in Fe-free medium, the abundance of some transcripts, such as those for genes encoding transporters and enzymes involved in Fe assimilation, change within minutes, before changes in intracellular Fe content are noticeable, suggestive of a sensitive mechanism for sensing Fe. Promoter reporter constructs indicate a transcriptional component to this immediate primary response. With acetate provided as a source of reduced carbon, transcripts encoding respiratory components are maintained relative to transcripts encoding components of photosynthesis and tetrapyrrole biosynthesis, indicating metabolic prioritization of respiration over photosynthesis. In contrast to the loss of chlorophyll, carotenoid content is maintained under Fe limitation despite a decrease in the transcripts for carotenoid biosynthesis genes, indicating carotenoid stability. These changes occur more slowly, only after the intracellular Fe quota responds, indicating a phased response in Chlamydomonas, involving both primary and secondary responses during acclimation to poor Fe nutrition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Engineered Membrane Vesicle Production via oprF or oprI Deletion Has Distinct Phenotypic Effects in Pseudomonas putida

Membrane vesicle (MV) production is a natural phenomenon in Gram-negative bacteria and represents an emerging synthetic biology tool for the secretion of biomolecules or bioproducts. Manipulation of membrane components has proven successful in enhancing MV production. However, the impact of membrane disruptions on strain fitness and protein composition warrants further investigation for the use of MVs in industrial bioprocesses. Here, we identify and characterize two genetic engineering strategies for inducing hypervesiculation-deletion of genes for the outer membrane porin OprF or the lipoprotein OprI-in the commonly used platform Pseudomonas putida KT2440. Deletion of oprI generated up to a 1.5-fold increase in MVs, larger MVs with a greater proportion of outer membrane proteins, and no significant impact on strain fitness compared to wild type. In contrast, deletion of oprF, relative to wild type, generated up to a 4-fold increase in MVs but diminished growth, permeabilized membranes, and increased cytosolic protein packaging. Both hypervesiculation phenotypes increased nontargeted and MV-targeted mNeonGreen extracellular signal by up to 6-fold, demonstrating vesiculation as a mechanism for protein secretion. Despite increased blebbing of MVs from gene deletions, proteins involved in membrane biosynthesis were not elevated relative to wild type. Overexpression of gpsA, which initiates glycerophospholipid biosynthesis, in the ΔoprF background improved the membrane integrity by 37% and maintained MV formation, highlighting the importance of membrane biosynthesis in restoring the membrane in hypervesiculating strains. Together, this study provides genetic engineering strategies with corresponding phenotypic outcomes toward providing a synthetic biology toolset for MV deployment in P. putida.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Gene and genome duplications have contrasting impacts on biosynthetic and flower developmental pathways in California poppy

Benzylisoquinoline alkaloids (BIAs) represent a vast group of specialized plant metabolites with diverse pharmaceutical applications, synthesized by a variety of gene families. Among the multiple plant lineages that produce BIAs, the most notable is the poppy family (Papaveraceae), with California poppy (Eschscholzia californica) emerging as a model organism. Here, we report a haplotype-resolved genome assembly, in combination with a high-density expression atlas, for California poppy. Genome analyses reveal recent diversification of BIA biosynthesis genes in poppy through localized duplications. Furthermore, we demonstrate that the degree of phylogenetic relatedness among paralogs within BIA biosynthesis-associated gene families correlates with similarities in gene expression. In contrast, gene families involved in carotenoid biosynthesis, which contributes to the intense orange petal pigmentation, are not phylogenetically clustered, and floral developmental regulators exhibit a high degree of retention of gene duplicates associated with ancient polyploidy events. These findings illustrate alternative roles for gene and genome duplications as drivers of trait evolution. Given the position of California poppy in the angiosperm phylogeny, the high-quality genomic resources generated for this work constitute a valuable resource for comparative genomic and transcriptomic analyses for poppies and flowering plants more generally.

Rössner, Le-Han [Justus-Liebig University, Giessen↗
DOE OSTI2026

Metabolic flux, metabolite, and transcript analysis uncover reprogramming of metabolism toward higher seed oil

Overexpression of WRINKLED1 (WRI1), a master regulator of glycolysis and fatty acid biosynthesis, together with DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1), which catalyzes the final step of triacylglycerol assembly, is a promising strategy for enhancing seed oil content. However, how these regulators coordinate system-wide metabolic reprogramming at the levels of gene expression, metabolite pools, and fluxes remains poorly understood. To address this, we performed 13 C-metabolic flux analysis, metabolomics, and transcriptomics on in vitro cultured pennycress (Thlaspi arvense L.) embryos overexpressing the native WRI1 and DGAT1 homologs. Here, in cultured embryos, WRI1/DGAT1 overexpression increased triacylglycerol accumulation by 28% while reducing protein content by 34%, relative to the wild type. Embryos showed ∼20-fold and 50-fold upregulation of WRI1 and DGAT1 along with induction of WRI1 target genes in glycolysis and fatty acid biosynthesis. Genes associated with photosynthesis and Calvin cycle functions were also upregulated, whereas genes encoding ribosomal proteins and seed storage proteins were strongly repressed, consistent with the observed lipid–protein tradeoff. Flux analysis revealed that enhanced triacylglycerol biosynthesis is supported by increased flux through the Rubisco shunt and cytosolic pyruvate kinase, while the oxidative pentose phosphate pathway and malic enzyme contributed little to NADPH or pyruvate supply. Metabolomic profiling revealed extensive perturbations in glycolytic intermediates, tricarboxylic acid cycle metabolites, and amino acids. In plant grown seeds, WRI1/DGAT1 lines also showed a modest but significant increase in total lipid content. Collectively, these findings reveal how WRI1 and DGAT1 reprogram central metabolism to enhance oil accumulation, with relevance to mature seeds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Switchable client specificity in a dual functional chaperone coordinates light-harvesting complex biogenesis

The proper assembly of light-harvesting complexes (LHCs) is critical for photosynthesis and requires the biogenesis of light-harvesting chlorophylla,b-binding proteins (LHCPs) to be coordinated with chlorophyll (Chl) biosynthesis. The mechanism underlying this coordination is not well understood. Here, we show that a conserved molecular chaperone, chloroplast signal recognition particle 43-kDa protein (cpSRP43), provides a molecular thermostat that helps maintain this coordination. cpSRP43 undergoes a conformational rearrangement between a well-folded closed state and a partially disordered open state. Closed cpSRP43 is dedicated to the biogenesis of LHCPs, whereas open cpSRP43 protects multiple Chl biosynthesis enzymes from heat-induced destabilization. Rising temperature shifts cpSRP43 to the open state, enabling it to protect heat-destabilized Chl biosynthesis enzymes. Our results reveal the molecular basis of a posttranslational mechanism for the thermoadaptation of LHC biogenesis. They also demonstrate how an adenosine triphosphate–independent chaperone uses conformational dynamics to switch its activity and client selectivity, thereby adapting to different proteostatic demands under shifting environmental conditions.

Science & Technology - Other Topics↗
DOE OSTI2024

An exopolysaccharide pathway from a freshwater Sphingomonas isolate

Bacteria embellish their cell envelopes with a variety of specialized polysaccharides. Biosynthesis pathways for these glycans are complex, and final products vary greatly in their chemical structures, physical properties, and biological activities. This tremendous diversity comes from the ability to arrange complex pools of monosaccharide building blocks into polymers with many possible linkage configurations. Due to the complex chemistry of bacterial glycans, very few biosynthetic pathways have been defined in detail. As part of an initiative to characterize novel polysaccharide biosynthesis enzymes, we isolated a bacterium from Lake Michigan called Sphingomonas sp. LM7 that is proficient in exopolysaccharide (EPS) production. We identified genes that contribute to EPS biosynthesis in LM7 by screening a transposon mutant library for colonies displaying altered colony morphology. A gene cluster was identified that appears to encode a complete wzy/wzx-dependent polysaccharide assembly pathway. Deleting individual genes in this cluster caused a non-mucoid phenotype and a corresponding loss of EPS secretion, confirming the role of this gene cluster in polysaccharide production. We extracted EPS from LM7 cultures and determined that it contains a linear chain of 3- and 4-linked glucose, galactose, and glucuronic acid residues. Finally, we show that the EPS pathway in Sphingomonas sp. LM7 diverges from that of sphingan-family EPSs and adhesive polysaccharides such as the holdfast that are present in other Alphaproteobacteria. Our approach of characterizing complete biosynthetic pathways holds promise for engineering polysaccharides with valuable properties.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Cross-family and phage-specific gene requirements for Klebsiella infection revealed by scalable RB-TnSeq genetic screens.

Bacteriophages are being cataloged at an accelerating pace and are recognized as key players in nutrient and energy cycling across ecosystems. Yet the bacterial genetic determinants that govern phage-host specificity and infection success remain poorly understood, particularly in clinically and ecologically important genera such as Klebsiella where prior receptor characterization has been almost entirely limited to capsulated strains. Here we used a randomly barcoded, genome-wide, loss-of-function transposon mutant library (RB-TnSeq) of Klebsiella sp. M5al, a naturally acapsular, nitrogen-fixing rhizobacterium, to generate the first systematic, cross-family map of phage receptor gene dependencies in Klebsiella. Challenging the library against 25 double-stranded DNA phages spanning five families in 213 parallel assays, we identified 42 bacterial genes associated with phage infection, of which 15 had no prior association with phage infection in any bacterial system. Disruption of surface receptor biosynthesis genes conferred cross-resistance across multiple phage families, while intracellular gene disruptions had predominantly phage-specific effects. Clonal validation of eight genes confirmed LPS outer core biosynthesis genes as primary receptor determinants alongside additional host factors spanning outer membrane transport, cofactor biosynthesis, and two-component signaling. Comparative analysis across all 25 phages revealed that phage genus rather than family is the stronger predictor of host gene dependency profiles, a finding with direct implications for the functional annotation of uncharacterized phage isolates and rational phage cocktail design. Together, these findings provide a community resource for linking phage genomic diversity to functional host interaction space in this ecologically and clinically important genus.

Gittrich, Marissa R↗
DOE OSTI2025

Sorghum bicolor BTx623 Nitrogen Grown Conditions Gene Expression Profiling

Dhurrin, a cyanogenic glucoside, plays an important role in Sorghum bicolor physiology and defense. The concentration of dhurrin in sorghum is influenced by both nitrogen status and stage of plant organ development. While nitrogen resupply activates the expression of genes for dhurrin biosynthesis, the molecular mechanisms underlying this regulation remain unclear. In this study, we investigated the transcriptional response of sorghum to nitrogen resupply following growth under nitrogen-limiting conditions. Using a time-course design, we measured hydrogen cyanide potential (HCNp), growth, and nitrate content at 0-, 2-, 6-, 12-, 24-, 36-, 48-, and 60-h after resupply and collected tissue for RNAseq analysis in parallel for analysis of gene expression and construction of gene regulatory networks (GRNs). HCNp (mg g−1 DW) increased significantly in leaf and stem tissues following nitrogen resupply, with increases in the leaf partially driven by continued declines in controls under ongoing nitrogen stress. Expression of the dhurrin pathway genes was upregulated in leaves from 24 h after nitrogen resupply, with diel expression patterns observable over the remaining time points. No upregulation was observed in roots or stems, suggesting that developmental context overrides environmental cues. GRN analysis identified candidate transcription factors regulating dhurrin biosynthesis genes, including members of the MYB, bZIP, and GARP-type transcription factor families. Some of these candidate transcription factors may be involved in relieving senescence-associated suppression of dhurrin biosynthesis and link nitrogen signaling to pathway activation. These findings provide new insight into the nitrogen-responsive regulation of dhurrin in sorghum, highlighting candidate regulators for future functional characterization.

cyanogenic glucoside↗
DOE OSTI2024

Sorghum bicolor BTx623 Nitrogen Grown Conditions Set2 Gene Expression Profiling

Dhurrin, a cyanogenic glucoside, plays an important role in Sorghum bicolor physiology and defense. The concentration of dhurrin in sorghum is influenced by both nitrogen status and stage of plant organ development. While nitrogen resupply activates the expression of genes for dhurrin biosynthesis, the molecular mechanisms underlying this regulation remain unclear. In this study, we investigated the transcriptional response of sorghum to nitrogen resupply following growth under nitrogen-limiting conditions. Using a time-course design, we measured hydrogen cyanide potential (HCNp), growth, and nitrate content at 0-, 2-, 6-, 12-, 24-, 36-, 48-, and 60-h after resupply and collected tissue for RNAseq analysis in parallel for analysis of gene expression and construction of gene regulatory networks (GRNs). HCNp (mg g−1 DW) increased significantly in leaf and stem tissues following nitrogen resupply, with increases in the leaf partially driven by continued declines in controls under ongoing nitrogen stress. Expression of the dhurrin pathway genes was upregulated in leaves from 24 h after nitrogen resupply, with diel expression patterns observable over the remaining time points. No upregulation was observed in roots or stems, suggesting that developmental context overrides environmental cues. GRN analysis identified candidate transcription factors regulating dhurrin biosynthesis genes, including members of the MYB, bZIP, and GARP-type transcription factor families. Some of these candidate transcription factors may be involved in relieving senescence-associated suppression of dhurrin biosynthesis and link nitrogen signaling to pathway activation. These findings provide new insight into the nitrogen-responsive regulation of dhurrin in sorghum, highlighting candidate regulators for future functional characterization.

cyanogenic glucoside↗
DOE OSTI2024

The crystal structure of Grindelia robusta 7,13-copalyl diphosphate synthase reveals active site features controlling catalytic specificity

Diterpenoid natural products serve critical functions in plant development and ecological adaptation and many diterpenoids have economic value as bioproducts. The family of class II diterpene synthases catalyzes the committed reactions in diterpenoid biosynthesis, converting a common geranylgeranyl diphosphate precursor into different bicyclic prenyl diphosphate scaffolds. Enzymatic rearrangement and modification of these precursors generate the diversity of bioactive diterpenoids. We report the crystal structure of Grindelia robusta 7,13-copalyl diphosphate synthase, GrTPS2, at 2.1 Å of resolution. GrTPS2 catalyzes the committed reaction in the biosynthesis of grindelic acid, which represents the signature metabolite in species of gumweed (Grindelia spp., Asteraceae). Grindelic acid has been explored as a potential source for drug leads and biofuel production. The GrTPS2 crystal structure adopts the conserved three-domain fold of class II diterpene synthases featuring a functional active site in the γβ-domain and a vestigial α-domain. Substrate docking into the active site of the GrTPS2 apo protein structure predicted catalytic amino acids. Biochemical characterization of protein variants identified residues with impact on enzyme activity and catalytic specificity. Specifically, mutagenesis of Y457 provided mechanistic insight into the position-specific deprotonation of the intermediary carbocation to form the characteristic 7,13 double bond of 7,13-copalyl diphosphate.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗