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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Chapter 6: An Improved Leaf Protoplast System for Highly Efficient Transient Expression in Switchgrass (Panicum virgatum L.)

As a robust perennial C4-type monocot plant and a native species to North America, switchgrass (Panicum virgatum) has been evaluated and designated as a strong candidate bioenergy crop by the U.S. DOE. Although genetic modifications of switchgrass have been used to successfully reduce the recalcitrance of switchgrass biomass for biofuel production, the generation of transgenic switchgrass is still a slow and laborious process. A transient protoplast system can provide an excellent platform to accelerate the selection of genes-of-interest for tailoring switchgrass biomass. However, partially due to the lack of the complete genomic information, the attempts to optimize the transient protoplast system for switchgrass remain scarce. In this chapter, we provide an improved protocol for switchgrass protoplast isolation, increased transformation efficiency using CsCl gradient ultracentrifugation-derived plasmid DNA and extended application of the transient switchgrass protoplast system to analyze protein expression using western blot.

BASIC BIOLOGICAL SCIENCES,BIOMASS FUELS↗
NASA NTRS1999

Model system for plant cell biology: GFP imaging in living onion epidermal cells

The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

NASA Discipline Plant Biology↗
DOE OSTI2025

In planta production of the nylon precursor beta-ketoadipate

Beta-ketoadipate (βKA) is an intermediate of the βKA pathway involved in the degradation of aromatic compounds in several bacteria and fungi. Beta-ketoadipate also represents a promising chemical for the manufacturing of performance-advantaged nylons. We established a strategy for the in planta synthesis of βKA via manipulation of the shikimate pathway and the expression of bacterial enzymes from the βKA pathway. Using Nicotiana benthamiana as a transient expression system, we demonstrated the efficient conversion of protocatechuate (PCA) to βKA when plastid-targeted bacterial-derived PCA 3,4-dioxygenase (PcaHG) and 3-carboxy-cis,cis-muconate cycloisomerase (PcaB) were co-expressed with 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroG) and 3-dehydroshikimate dehydratase (QsuB). This metabolic pathway was reconstituted in Arabidopsis by introducing a construct (pAtβKA) with stacked pcaG, pcaH, and pcaB genes into a PCA-overproducing genetic background that expresses AroG and QsuB (referred as QsuB-2). The resulting QsuB-2 x pAtβKA stable lines displayed βKA titers as high as 0.25 % on a dry weight basis in stems, along with a drastic reduction in lignin content and improvement of biomass saccharification efficiency compared to wild-type controls, and without any significant reduction in biomass yields. Using biomass sorghum as a potential crop for large-scale βKA production, techno-economic analysis indicated that βKA accumulated at titers of 0.25 % and 4 % on a dry weight basis could be competitively priced in the range of $2.04-34.49/kg and $0.47-2.12/kg, respectively, depending on the selling price of the residual biomass recovered after βKA extraction. This study lays the foundation for a more environmentally-friendly synthesis of βKA using plants as production hosts.

Kazaz, Sami↗
DOE OSTI2020

Use of virus‐induced gene silencing to characterize genes involved in modulating hypersensitive cell death in maize

Abstract Plant disease resistance proteins (R‐proteins) detect specific pathogen‐derived molecules, triggering a defence response often including a rapid localized cell death at the point of pathogen penetration called the hypersensitive response (HR). The maize Rp1‐D21 gene encodes a protein that triggers a spontaneous HR causing spots on leaves in the absence of any pathogen. Previously, we used fine mapping and functional analysis in a Nicotiana benthamiana transient expression system to identify and characterize a number of genes associated with variation in Rp1‐D21 ‐induced HR. Here we describe a system for characterizing genes mediating HR, using virus‐induced gene silencing (VIGS) in a maize line carrying Rp1‐D21 . We assess the roles of 12 candidate genes. Three of these genes, SGT1 , RAR1 , and HSP90 , are required for HR induced by a number of R‐proteins across several plant–pathogen systems. We confirmed that maize HSP90 was required for full Rp1‐D21 ‐induced HR. However, suppression of SGT1 expression unexpectedly increased the severity of Rp1‐D21 ‐induced HR while suppression of RAR1 expression had no measurable effect. We confirmed the effects on HR of two genes we had previously validated in the N. benthamiana system, hydroxycinnamoyltransferase and caffeoyl CoA O‐methyltransferase . We further showed the suppression the expression of two previously uncharacterized, candidate genes, IQ calmodulin binding protein ( IQM3 ) and vacuolar protein sorting protein 37 , suppressed Rp1‐D21 ‐induced HR. This approach is an efficient way to characterize the roles of genes modulating the hypersensitive defence response and other dominant lesion phenotypes in maize.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A split ribozyme system for in vivo plant RNA imaging and genetic engineering

RNA plays a central role in plants, governing various cellular and physiological processes. Monitoring its dynamic abundance provides a discerning understanding of molecular mechanisms underlying plant responses to internal (developmental) and external (environmental) stimuli, paving the way for advances in plant biotechnology to engineer crops with improved resilience, quality and productivity. In general, traditional methods for analysis of RNA abundance in plants require destructive, labour-intensive and time-consuming assays. To overcome these limitations, we developed a transformative innovation for in vivo RNA imaging in plants. Specifically, we established a synthetic split ribozyme system that converts various RNA signals to orthogonal protein outputs, enabling in vivo visualisation of various RNA signals in plants. We demonstrated the utility of this system in transient expression experiments (i.e., leaf infiltration in Nicotiana benthamiana ) to detect RNAs derived from transgenes and tobacco rattle virus, respectively. Also, we successfully engineered a split ribozyme-based biosensor in Arabidopsis thaliana for in vivo visualisation of endogenous gene expression at the cellular level, demonstrating the feasibility of multi-scale (e.g., cellular and tissue level) RNA imaging in plants. Furthermore, we developed a platform for easy incorporation of different protein outputs, allowing for flexible choice of reporters to optimise the detection of target RNAs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Grid-Supportive Loads - A New Approach to Increasing Renewable Energy in Power Systems

This paper demonstrates the potential of inverter-based loads to support grid reliability during power system transients thereby enabling reliable integration of renewable energy in power systems. Such loads are referred to in this paper as grid-supportive loads (GSLs). A new GSL model is developed that simulates the transient response capabilities that can be programmed in electronic loads. The model’s design enables it to be easily integrated in widely used commercial power system transient analysis software. Theoretical expressions are derived that explain the workings of the GSL model. The performance, numerical stability, and impact of the GSL model is validated on 9-bus and 2000-bus synthetic power system models using generator tripping and bus fault disturbances. Results on the 2000 bus system show that in the absence of frequency support from wind/solar generation resources, just 20% of loads with grid-supportive capabilities can improve frequency response by up to 2000 MW/0.1 Hz and reduce deviation in frequency at nadir by up to 60% compared to the situation when GSLs are absent. Power system reliability also improves under fault events. Here, it is further shown that GSLs can aid in integrating more renewable generation without degrading the overall transient response of the power system.

24 POWER TRANSMISSION AND DISTRIBUTION↗
NASA NTRS1991

Analytical and experimental study of vibrations in a gear transmission

An analytical simulation of the dynamics of a gear transmission system is presented and compared to experimental results from a gear noise test rig at the NASA Lewis Research Center. The analytical procedure developed couples the dynamic behaviors of the rotor-bearing-gear system with the response of the gearbox structure. The modal synthesis method is used in solving the overall dynamics of the system. Locally each rotor-gear stage is modeled as an individual rotor-bearing system using the matrix transfer technique. The dynamics of each individual rotor are coupled with other rotor stages through the nonlinear gear mesh forces and with the gearbox structure through bearing support systems. The modal characteristics of the gearbox structure are evaluated using the finite element procedure. A variable time steping integration routine is used to calculate the overall time transient behavior of the system in modal coordinates. The global dynamic behavior of the system is expressed in a generalized coordinate system. Transient and steady state vibrations of the gearbox system are presented in the time and frequency domains. The vibration characteristics of a simple single mesh gear noise test rig is modeled. The numerical simulations are compared to experimental data measured under typical operating conditions. The comparison of system natural frequencies, peak vibration amplitudes, and gear mesh frequencies are generally in good agreement.

Choy, F. K.↗
DOE OSTI2022

Protoplast-Based Transient Expression and Gene Editing in Shrub Willow ( Salix purpurea L .)

Shrub willows (Salix section Vetrix) are grown as a bioenergy crop in multiple countries and as ornamentals across the northern hemisphere. To facilitate the breeding and genetic advancement of shrub willow, there is a strong interest in the characterization and functional validation of genes involved in plant growth and biomass production. While protocols for shoot regeneration in tissue culture and production of stably transformed lines have greatly advanced this research in the closely related genus Populus, a lack of efficient methods for regeneration and transformation has stymied similar advancements in willow functional genomics. Moreover, transient expression assays in willow have been limited to callus tissue and hairy root systems. Here we report an efficient method for protoplast isolation from S. purpurea leaf tissue, along with transient overexpression and CRISPR-Cas9 mediated mutations. This is the first such report of transient gene expression in Salix protoplasts as well as the first application of CRISPR technology in this genus. These new capabilities pave the way for future functional genomics studies in this important bioenergy and ornamental crop.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2011

System Re-set: High LET Radiation or Transient Musculoskeletal Disuse Cause Lasting Changes in Oxidative Defense Pathways Within Bone

Six months post-IR, there were no notable changes in skeletal expression of 84 principal genes in the p53 signaling pathway due to low dose IR (0.5Gy), HU, or both. In contrast, numerous genes relevant to oxidative stress were regulated by the treatments, typically in a direction indicative of increased oxidative stress and impaired defense. IR and HU independently reduced (between 0.46 to 0.88 fold) expression levels of Noxa1, Gpx3, Prdx2, Prdx3, and Zmynd17. Surprisingly, transient HU alone (sham-irradiated) decreased expression of several redox-related genes (Gpx1,Gstk1, Prdx1, Txnrd2), which were not affected significantly by IR alone. Irradiation increased (1.13 fold) expression of a gene responsible for production of superoxides by neutrophils (NCF2). Of interest, only combined treatment with HU and IR led to increased expression levels of Ercc2, (1.19 fold), a DNA excision repair enzyme. Differences in gene expression levels may reflect a change in gene expression on a per cell basis, a shift in the repertoire of specific cell types within the tissue, or both. Serum nitrite/nitrate levels were elevated to comparable levels (1.6-fold) due to IR, HU or both, indicative of elevated systemic nitrosyl stress. CONCLUSIONS The magnitude of changes in skeletal expression of oxidative stress-related genes six months after irradiation and/or transient unloading tended to be relatively modest (0.46-1.15 fold), whereas the p53 pathway was not affected. The finding that many different oxidative stress-related genes differed from controls at this late time point implicates a generalized impairment of oxidative defense within skeletal tissue, which coincides with both profound radiation damage to osteoprogenitors/stem cells in bone marrow and impaired remodeling of mineralized tissue.

Kumar, Akhilesh↗
NASA NTRS1973

Nonlinear transient analysis of multi-mass flexible rotors - theory and applications

The equations of motion necessary to compute the transient response of multi-mass flexible rotors are formulated to include unbalance, rotor acceleration, and flexible damped nonlinear bearing stations. A method of calculating the unbalance response of flexible rotors from a modified Myklestad-Prohl technique is discussed in connection with the method of solution for the transient response. Several special cases of simplified rotor-bearing systems are presented and analyzed for steady-state response, stability, and transient behavior. These simplified rotor models produce extensive design information necessary to insure stable performance to elastic mounted rotor-bearing systems under varying levels and forms of excitation. The nonlinear journal bearing force expressions derived from the short bearing approximation are utilized in the study of the stability and transient response of the floating bush squeeze damper support system. Both rigid and flexible rotor models are studied, and results indicate that the stability of flexible rotors supported by journal bearings can be greatly improved by the use of squeeze damper supports. Results from linearized stability studies of flexible rotors indicate that a tuned support system can greatly improve the performance of the units from the standpoint of unbalanced response and impact loading. Extensive stability and design charts may be readily produced for given rotor specifications by the computer codes presented in this analysis.

Kirk, R. G.↗
DOE OSTI2026

Causes and consequences of experimental variation in Nicotiana benthamiana transient expression

Infiltration of Agrobacterium tumefaciens into Nicotiana benthamiana has become a foundational technique in plant biology, enabling efficient delivery of transgenes in planta with technical ease, robust signal, and relatively high throughput. Despite transient expression’s prevalence in disciplines such as synthetic biology, little work has been done to describe and address the variability inherent in this system, a concern for experiments that rely on highly quantitative readouts. In a comprehensive analysis of N. benthamiana agroinfiltration experiments, we model sources of variability that affect transient expression. Our findings emphasize the need to validate normalization methods under the specific conditions of each study, as distinct normalization schemes do not always reduce variation either within or between experiments. Using a dataset of 1915 plants collected over three years, we develop a model of variation in N. benthamiana transient expression, using power analysis to determine the number of individual plants required for a given effect size. Drawing on our longitudinal data, these findings inform practical guidelines for minimizing variability through strategic experimental design and power analysis, providing a foundation for more robust and reproducible use of N. benthamiana in quantitative plant biology and synthetic biology applications.

Tang, Sophia N. [Joint BioEnergy Institute (JBEI),↗
NASA NTRS1955

Analog Study of Interacting and Noninteracting Multiple-loop Control Systems for Turbojet Engines

The results of an analog investigation of several turbojet-engine control configurations is presented in this report. Both proportional and proportional-plus-integral controllers were studied, and compensating terms for engine interaction were added to the control system. Data were obtained on the stability limits and the transient responses of these various configurations. Analytical expressions in terms of the component transfer functions were developed for the configurations studied, and the optimum form for the compensation terms was determined. It was found that the addition of the integral term, while making the system slower and more oscillatory, was desirable in that it made the final values of the system parameters independent of source of disturbance and also eliminated droop in these parameters. Definite improvement in system characteristics resulted from the use of proper compensation terms. At comparable gain points the compensated system was faster and more stable. Complete compensation eliminated engine interaction, permitting each loop to be developed to an optimum point independently.

Pack, George J↗
NASA NTRS1983

Transient nucleation in condensed systems

Using classical nucleation theory we consider transient nucleation occurring in a one-component, condensed system under isothermal conditions. We obtain an exact closed-form expression for the time dependent cluster populations. In addition, a more versatile approach is developed: a numerical simulation technique which models directly the reactions by which clusters are produced. This simulation demonstrates the evolution of cluster populations and nucleation rate in the transient regime. Results from the simulation are verified by comparison with exact analytical solutions for the steady state. Experimental methods for measuring transient nucleation are assessed, and it is demonstrated that the observed behavior depends on the method used. The effect of preexisting cluster distributions is studied. Previous analytical and numerical treatments of transient nucleation are compared to the solutions obtained from the simulation. The simple expressions of Kashchiev are shown to give good descriptions of the nucleation behavior.

Kelton, K. F.↗
NASA NTRS1993

Mixing and transient interface condensation of a liquid hydrogen tank

Experiments were conducted to investigate the effect of axial jet-induced mixing on the pressure reduction of a thermally stratified liquid hydrogen tank. The tank was nearly cylindrical, having a volume of about 0.144 cu m with 0.559 m in diameter and 0.711 m length. A mixer/pump unit, which had a jet nozzle outlet of 0.0221 m in diameter was located 0.178 m from the tank bottom and was installed inside the tank to generate the axial jet mixing and tank fluid circulation. Mixing tests began with the tank pressures at which the thermal stratification results in 4.9-6.2 K liquid subcooling. The mixing time and transient vapor condensation rate at the liquid-vapor interface are determined. Two mixing time correlations, based on the thermal equilibrium and pressure equilibrium, are developed and expressed as functions of system and buoyancy parameters. The limited liquid hydrogen data of the present study shows that the modified steady state condensation rate correlation may be used to predict the transient condensation rate in a mixing process if the instantaneous values of jet sub cooling and turbulence intensity at the interface are employed.

Lin, C. S.↗
DOE OSTI2023

Auger spectroscopy beyond the ultra-short core-hole relaxation time approximation

Abstract We present a time-dependent computational approach to study Auger electron spectroscopy (AES) beyond the ultra-short core-hole relaxation time approximation and, as a test case, we apply it to the paradigmatic example of a one-dimensional Mott insulator represented by a half-filled Hubbard chain. The Auger spectrum is usually calculated by assuming that, after the creation of a core-hole, the system thermalizes almost instantaneously. This leads to a relatively simple analytical expression that uses the ground-state with a core-hole as a reference state and ignores all the transient dynamics related to the screening of the core-hole. In this picture, the response of the system can be associated to the pair spectral function. On the other hand, in our numerical calculations, the core hole is created by a light pulse, allowing one to study the transient dynamics of the system in terms of the pulse duration and in the non-perturbative regime. Time-dependent density matrix renormalization group calculations reveal that the relaxation process involves the creation of a polarization cloud of doublon excitations that have an effect similar to photo-doping. As a consequence, there is a leak of spectral weight to higher energies into what otherwise would be the Mott gap. For longer pulses, these excited states, mostly comprised of doublons, can dominate the spectrum. By changing the duration of the light-pulse, the entire screening process can be resolved in time.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
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↗
NASA NTRS1999

Rapid and systemic accumulation of chloroplast mRNA-binding protein transcripts after flame stimulus in tomato

It has been shown that tomato (Lycopersicon esculentum) plants respond to flame wounding and electrical stimulation by a rapid (15 min) and systemic up-regulation of proteinase inhibitor (pin) genes. To find other genes having a similar expression pattern, we used subtractive cDNA screening between flamed and control plants to select clones up-regulated by flame wounding. We report the characterization of one of them, a chloroplast mRNA-binding protein encoded by a single gene and expressed preferentially in the leaves. Systemic gene expression in response to flaming in the youngest terminal leaf exhibited three distinct phases: a rapid and transient increase (5-15 min) in transcript accumulation, a decline to basal levels (15-45 min), and then a second, more prolonged increase (60-90 min). In contrast, after a mechanical wound the rapid, transient increase (5 min) was followed by a rapid decline to basal levels but no later, prolonged accumulation. In the petiole, the initial flame-wound-evoked transient increase (15 min) was followed by a continuous decline for 3 h. The nature of the wound signal(s) causing such rapid changes in transcript abundance is discussed in relation to electrical signaling, which has recently been implicated in plant responses to wounding.

NASA Discipline Plant Biology↗
NASA NTRS2008

Numerical Determination of Critical Conditions for Thermal Ignition

The determination of ignition or thermal explosion in an oxidizing porous body of material, as described by a dimensionless reaction-diffusion equation of the form .tu = .2u + .e-1/u over the bounded region O, is critically reexamined from a modern perspective using numerical methodologies. First, the classic stationary model is revisited to establish the proper reference frame for the steady-state solution space, and it is demonstrated how the resulting nonlinear two-point boundary value problem can be reexpressed as an initial value problem for a system of first-order differential equations, which may be readily solved using standard algorithms. Then, the numerical procedure is implemented and thoroughly validated against previous computational results based on sophisticated path-following techniques. Next, the transient nonstationary model is attacked, and the full nonlinear form of the reaction-diffusion equation, including a generalized convective boundary condition, is discretized and expressed as a system of linear algebraic equations. The numerical methodology is implemented as a computer algorithm, and validation computations are carried out as a prelude to a broad-ranging evaluation of the assembly problem and identification of the watershed critical initial temperature conditions for thermal ignition. This numerical methodology is then used as the basis for studying the relationship between the shape of the critical initial temperature distribution and the corresponding spatial moments of its energy content integral and an attempt to forge a fundamental conjecture governing this relation. Finally, the effects of dynamic boundary conditions on the classic storage problem are investigated and the groundwork is laid for the development of an approximate solution methodology based on adaptation of the standard stationary model.

Luo, W.↗