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At least 1,135 records · Page 63
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Implementation of a Practical Teaching Course on Protein Engineering

Protein Engineering is a highly evolved field of engineering aimed at developing proteins for specific industrial, medical, and research applications. Here, we present a practical teaching course to demonstrate fundamental techniques used to express, purify and analyze a recombinant protein produced in Escherichia coli—the enhanced green fluorescent protein (eGFP). The methodologies used for eGFP production were introduced sequentially over six laboratory sessions and included (i) bacterial growth, (ii) sonication (for cell lysis), (iii) affinity chromatography and dialysis (for eGFP purification), (iv) bicinchoninic acid (BCA) and fluorometry assays for total protein and eGFP quantification, respectively, and (v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for qualitative analysis. All groups were able to isolate the eGFP from the cell lysate with purity levels up to 72%. Additionally, a mass balance analysis performed by the students showed that eGFP yields up to 46% were achieved at the end of the purification process following the adopted procedures. A sensitivity analysis was performed to pinpoint the most critical steps of the downstream processing.

Gomes, Luciana (ORCID:0000000289921097)↗
DOE OSTI2020

Groundwater Depth Overrides Tree-Species Effects on the Structure of Soil Microbial Communities Involved in Nitrogen Cycling in Plantation Forests

Microbial communities found in soil ecosystems play important roles in decomposing organic materials and recycling nutrients. A clear understanding on how biotic and abiotic factors influence the microbial community and its functional role in ecosystems is fundamental to terrestrial biogeochemistry and plant production. The purpose of this study was to investigate microbial communities and functional genes involved in nitrogen cycling as a function of groundwater depth (deep and shallow), tree species (pine and eucalypt), and season (spring and fall). Soil fungal, bacterial, and archaeal communities were determined by length heterogeneity polymerase chain reaction (LH-PCR). Soil ammonia oxidation archaeal (AOA) amoA gene, ammonia oxidation bacterial (AOB) amoA gene, nitrite oxidoreductase nrxA gene, and denitrifying bacterial narG, nirK, nirS, and nosZ genes were further studied using PCR and denaturing gradient gel electrophoresis (DGGE). Soil fungal and bacterial communities remained similar between tree species and groundwater depths, respectively, regardless of season. Soil archaeal communities remained similar between tree species but differed between groundwater depths in the spring only. Archaeal amoA for nitrification and bacterial nirK and nosZ genes for denitrification were detected in DGGE, whereas bacterial amoA and nrxA for nitrification and bacterial narG and nirS genes for denitrification were undetectable. The detected nitrification and denitrification communities varied significantly with groundwater depth. There was no significant difference of nitrifying archaeal amoA or denitrifying nirK communities between different tree species regardless of season. The seasonal difference in microbial communities and functional genes involved in nitrogen cycling suggests microorganisms exhibit seasonal dynamics that likely impact relative rates of nitrification and denitrification.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Effect of Cationic (Na + ) and Anionic (F – ) Co-Doping on the Structural and Electrochemical Properties of LiNi 1/3 Mn 1/3 Co 1/3 O 2 Cathode Material for Lithium-Ion Batteries

Elemental doping for substituting lithium or oxygen sites has become a simple and effective technique to improve the electrochemical performance of layered cathode materials. Compared with single-element doping, this work presents an unprecedented contribution to the study of the effect of Na + /F – co-doping on the structure and electrochemical performance of LiNi 1/3 Mn 1/3 Co 1/3 O 2 . The co-doped Li 1-z Na z Ni 1/3 Mn 1/3 Co 1/3 O 2-z F z (z = 0.025) and pristine LiNi 1/3 Co 1/3 Mn 1/3 O 2 materials were synthesized via the sol–gel method using EDTA as a chelating agent. Structural analyses, carried out by X-ray diffraction, Raman spectroscopy, and X-ray photoelectron spectroscopy, revealed that the Na + and F – dopants were successfully incorporated into the Li and O sites, respectively. The co-doping resulted in larger Li-slab spacing, a lower degree of cation mixing, and the stabilization of the surface structure, which substantially enhanced the cycling stability and rate capability of the cathode material. The Na/F co-doped LiNi 1/3 Mn 1/3 Co 1/3 O 2 electrode delivered an initial specific capacity of 142 mAh g –1 at a 1C rate (178 mAh g –1 at 0.1C), and it maintained 50% of its initial capacity after 1000 charge–discharge cycles at a 1C rate.

25 ENERGY STORAGE↗
DOE OSTI2023

Nuclear Magnetic Resonance Relaxation Pathways in Electrolytes for Energy Storage

Nuclear Magnetic Resonance (NMR) spin relaxation times have been an instrumental tool in deciphering the local environment of ionic species, the various interactions they engender and the effect of these interactions on their dynamics in conducting media. Of particular importance has been their application in studying the wide range of electrolytes for energy storage, on which this review is based. Here we highlight some of the research carried out on electrolytes in recent years using NMR relaxometry techniques. Specifically, we highlight studies on liquid electrolytes, such as ionic liquids and organic solvents; on semi-solid-state electrolytes, such as ionogels and polymer gels; and on solid electrolytes such as glasses, glass ceramics and polymers. Although this review focuses on a small selection of materials, we believe they demonstrate the breadth of application and the invaluable nature of NMR relaxometry.

25 ENERGY STORAGE↗
DOE OSTI2025

Synthesis and Lithium Storage Properties of Spinel (Al 0.2 Mn 0.2 Co 0.2 Ni 0.2 Zn 0.2 ) 3 O 4 High-Entropy Oxide

High-entropy oxides (HEOs) have garnered significant interest as next-generation anode materials for lithium-ion batteries (LIBs) due to their high theoretical specific capacity and excellent structural stability. This study successfully synthesized spinel-structured (Al 0.2 Mn 0.2 Co 0.2 Ni 0.2 Zn 0.2 ) 3 O 4 HEO via a sol–gel method. The material was characterized by XRD, Raman and TEM, confirming a homogeneous single-phase spinel structure, with uniformly distributed elements-a hallmark of HEOs. Electrochemical tests demonstrated a stable cycling performance (438 mAh g −1 at 100 mA g −1 after 100 cycles and 350 mAh g −1 at 1 A g −1 after 1000 cycles) and rate capacity of 159 mAh g −1 at 2 A g −1 , The remarkable long-term cyclability and good rate capability highlight the potential of this HEO for practical applications in durable, high-power lithium-ion batteries. This work underscores the advantage of incorporating structurally stabilizing elements in HEOs for advanced energy storage.

anode materials↗
DOE OSTI2024

Characterization of Levan Fructan Produced by a Gluconobacter japonicus Strain Isolated from a Sugarcane Processing Facility

During raw sugarcane processing, a significant portion of lost sucrose is attributable to microbial degradation. Sucrose consumption by many bacteria is also linked to the production of exopolysaccharides (EPS) such as dextrans and fructans. These resulting EPS cause operational challenges during raw sugar manufacturing. Here, we report the characterization of EPS from a fructan-forming Gluconobacter japonicus bacterium that we previously isolated from a Louisiana sugarcane factory. The genome sequencing revealed the presence of two encoded levansucrase genes, lsrA and lsrB. One levansucrase, LsrB, was detected in the secreted protein fraction of G. japonicus LASM12 by QTOF LC-MS. The spotting assays indicated that G. japonicus produces EPS using sucrose and raffinose as substrates. The G. japonicus EPS correlated with levan fructan commercial standards by 1H-NMR, and with the characteristic carbohydrate fingerprint region for FTIR spectra, confirming that the G. japonicus EPS is levan fructan. The glycosyl composition and glycosyl linkage analysis revealed a linear β-2,6-fructofuranosyl polysaccharide with occasional (5.7%) β-2,1-fructofuranosyl branches. The gel permeation chromatography of the levan fructan EPS showed two main peaks at 4.5 kDa and 8 kDa and a very minor peak at 500 kDa. G. japonicus was identified as a producer of levan fructan. These findings will be useful for future studies aimed at evaluating the impact of levan fructans on sugar crop processing, which have been historically underestimated in industry.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Chloropyridinyl Esters of Nonsteroidal Anti-Inflammatory Agents and Related Derivatives as Potent SARS-CoV-2 3CL Protease Inhibitors

We report the design and synthesis of a series of new 5-chloropyridinyl esters of salicylic acid, ibuprofen, indomethacin, and related aromatic carboxylic acids for evaluation against SARS-CoV-2 3CL protease enzyme. These ester derivatives were synthesized using EDC in the presence of DMAP to provide various esters in good to excellent yields. Compounds are stable and purified by silica gel chromatography and characterized using 1 H-NMR, 13 C-NMR, and mass spectral analysis. These synthetic derivatives were evaluated in our in vitro SARS-CoV-2 3CLpro inhibition assay using authentic SARS-CoV-2 3CLpro enzyme. Compounds were also evaluated in our in vitro antiviral assay using quantitative VeroE 6 cell-based assay with RNAqPCR. A number of compounds exhibited potent SARS-CoV-2 3CLpro inhibitory activity and antiviral activity. Compound 9a was the most potent inhibitor, with an enzyme IC 50 value of 160 nM. Compound 13b exhibited an enzyme IC 50 value of 4.9 µM. However, it exhibited a potent antiviral EC 50 value of 24 µM in VeroE6 cells. Remdesivir, an RdRp inhibitor, exhibited an antiviral EC 50 value of 2.4 µM in the same assay. We assessed the mode of inhibition using mass spectral analysis which suggested the formation of a covalent bond with the enzyme. To obtain molecular insight, we have created a model of compound 9a bound to SARS-CoV-2 3CLpro in the active site.

3CLpro inhibitors↗
DOE OSTI2020

Styrene-Based Elastomer Composites with Functionalized Graphene Oxide and Silica Nanofiber Fillers: Mechanical and Thermal Conductivity Properties

The mechanical and thermal conductivity properties of two composite elastomers were studied. Styrene–butadiene rubber (SBR) filled with functionalized graphene oxide (GO) and silica nanofibers, and styrene–butadiene–styrene (SBS) block copolymers filled with graphene oxide. For the SBR composites, GO fillers with two different surface functionalities were synthesized (cysteamine and dodecylamine) and dispersed in the SBR using mechanical and liquid mixing techniques. The hydrophilic cysteamine-based GO fillers were dispersed in the SBR by mechanical mixing, whereas the hydrophobic dodecylamine-based GO fillers were dispersed in the SBR by liquid mixing. Silica nanofibers (SnFs) were fabricated by electrospinning a sol–gel precursor solution. The surface chemistry of the functionalized fillers was studied in detail. The properties of the composites and the synergistic improvements between the GO and SnFs are presented. For the SBS composites, GO fillers were dispersed in the SBS elastomer at several weight percent loadings using liquid mixing. Characterization of the filler material and the composite elastomers was performed using x-ray photoelectron spectroscopy, x-ray diffraction, transmission electron microscopy, scanning electron microscopy, thermogravimetric analysis, dynamic mechanical analysis, tensile testing, nanoindentation, thermal conductivity and abrasion testing.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Hybrid Mesoporous Carbon/Copper Ferrite Electrode for Asymmetric Supercapacitors

Asymmetric supercapacitors (ASCs) with two dissimilar electrodes are known to exhibit relatively moderate energy and power densities. If electrodes derived from earth-abundant materials or renewable resources such as lignocellulosic biomass (LCB) are used for fabrication, energy storage systems are expected to become less expensive and more sustainable. Hybrid electrode materials have advantages such as higher surface area, better chemical stability, and superior energy density. This study reports on the synthesis of a novel hybrid electrode material containing porous carbon (POC) and copper ferrite, which is designated as POC@Cu-ferrite, and its electrochemical performance in ASC configuration. Corn stover derived hydrochar is utilized for the sol–gel synthesis of POC@Cu-ferrite hybrid material using earth-abundant Cu and Fe-based precursors. This material is characterized using X-ray diffraction (XRD), Raman spectroscopy, Brunauer–Emmett–Teller (BET) surface area analyzer, and scanning and transmission electron microscopy (SEM/TEM). As-synthesized Cu-ferrite is found to contain 89.2% CuFe 2 O 4 and 10.8% Fe 2 O 3 , whereas other phases such as Fe 3 O 4 , CuFeO 2 , and CuO are observed for the POC@Cu-ferrite. BET-specific surface area (SSA) and pore volume of POC@Cu-ferrite are observed as 1068 m 2 /g and 0.72 cm 3 /g, respectively. POC@Cu-ferrite hybrid electrode is used with POC opposite electrode to fabricate ASC, which is tested using Gamry G-300 potentiostat/galvanostat/ZRA to obtain cyclic voltammetry (CV) profiles and galvanostatic charge–discharge (GCD) plots. ASC is also prepared using Cu-ferrite and POC materials and its specific capacitance and stability are compared with ASCs prepared with POC@Cu-ferrite and POC or graphene nanoplatelets (GNPs) electrodes. POC@Cu-ferrite hybrid electrode is found to be superior with a 2-fold higher capacitance and significant electrochemical stability over 100 GCD cycles as compared to the Cu-ferrite electrode.

36 MATERIALS SCIENCE↗
DOE OSTI2021

3D Printing of High Viscosity Reinforced Silicone Elastomers

Recent advances in additive manufacturing, specifically direct ink writing (DIW) and ink-jetting, have enabled the production of elastomeric silicone parts with deterministic control over the structure, shape, and mechanical properties. These new technologies offer rapid prototyping advantages and find applications in various fields, including biomedical devices, prosthetics, metamaterials, and soft robotics. Stereolithography (SLA) is a complementary approach with the ability to print with finer features and potentially higher throughput. However, all high-performance silicone elastomers are composites of polysiloxane networks reinforced with particulate filler, and consequently, silicone resins tend to have high viscosities (gel- or paste-like), which complicates or completely inhibits the layer-by-layer recoating process central to most SLA technologies. Herein, the design and build of a digital light projection SLA printer suitable for handling high-viscosity resins is demonstrated. Further, a series of UV-curable silicone resins with thiol-ene crosslinking and reinforced by a combination of fumed silica and MQ resins are also described. The resulting silicone elastomers are shown to have tunable mechanical properties, with 100–350% elongation and ultimate tensile strength from 1 to 2.5 MPa. Three-dimensional printed features of 0.4 mm were achieved, and complexity is demonstrated by octet-truss lattices that display negative stiffness.

36 MATERIALS SCIENCE↗
DOE OSTI2022

Synthesis and Characterization of Copolymers and Nanocomposites from Limonene, Styrene and Organomodified-Clay Using Ultrasonic Assisted Method

In the present work, we report a simple synthesis method for preparation of copolymers and nanocomposites from limonene and styrene using clay as a catalyst. The copolymerization reaction is carried out by using a proton exchanged clay as a catalyst called Mag-H+. The effect of temperature, reaction time and amount of catalyst were studied, and the obtained copolymer structure (lim-co-sty) is characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (1H-NMR) and differential scanning calorimetry (DSC). The molecular weight of the obtained copolymer is determined by gel permeation chromatography (GPC) and is about 4500 g·mol−1. The (lim-co-sty/Mag 1%, 3%, 7% and 10% by weight of clay) nanocomposites were prepared through polymer/clay mixture in solution method using ultrasonic irradiation, in the presence of Mag-CTA+ as green nano-reinforcing filler. The Mag-CTA+ is organophilic silicate clay prepared through a direct exchange process, using cetyltrimethylammonuim bromide (CTAB). The prepared lim-co-sty/Mag nanocomposites have been extensively characterized by FT-IR spectroscopy, X-ray diffraction (XRD), scanning electronic microscopy (SEM) and transmission electronic microscopy (TEM). TEM analysis confirms the results obtained by XRD and clearly show that the obtained nanocomposites are partially exfoliated for the lower amount of clay (1% and 3% wt) and intercalated for higher amounts of clay (7% and 10% wt). Moreover, thermogravimetric analysis (TGA) indicated an enhancement of thermal stability of nanocomposites compared with the pure copolymer.

Derdar, Hodhaifa↗
DOE OSTI2025

Enabling Industrial Re-Use of Large-Format Additive Manufacturing Molding and Tooling

Large-format additive manufacturing (LFAM) is an enabling manufacturing technology capable of producing large parts with highly complex geometries for a wide variety of applications, including automotive, infrastructure/construction, and aerospace mold and tooling. In the past decade, the LFAM industry has seen widespread use of bio-based, glass, and/or carbon fiber reinforced thermoplastic composites which, when printed, serve as a lower-cost alternative to metallic parts. One of the highest-volume materials utilized by the industry is carbon fiber (CF)-filled polycarbonate (PC), which in out-of-autoclave applications can achieve comparable mechanical performance to metal at a significantly lower cost. Previous work has shown that if this material is recovered at various points throughout the manufacturing process for both the lab and pilot scale, it can be mechanically recycled with minimal impacts on the functional performance and printability of the material while significantly reducing the feedstock costs. End-of-life (EOL) CF-PC components were processed through industrial shredding, melt compounding, and LFAM equipment, followed by evaluation of the second-life material properties. Experimental assessments included quantitative analysis of fiber length attrition, polymer molecular weight degradation using gel permeation chromatography (GPC), density changes via pycnometry, thermal performance using dynamic mechanical analysis (DMA), and mechanical performance (tensile properties) in both the X- and Z-directions. Results demonstrated a 24.6% reduction in average fiber length compared to virgin prints, accompanied by a 21% decrease in X-direction tensile strength and a 39% reduction in tensile modulus. Despite these reductions, Z-direction tensile modulus improved by 4%, density increased by 6.8%, and heat deflection temperature (HDT) under high stress retained over 97% of its original value. These findings underscore the potential for integrating mechanically recycled CF-PC into industrial LFAM applications while highlighting the need for technological innovations to mitigate fiber degradation and enhance material performance for broader adoption. This critical step toward circular material practices in LFAM offers a pathway to reducing feedstock costs and environmental impact while maintaining functional performance in industrial applications.

additive manufacturing↗
DOE OSTI2020

Bent DNA Bows as Sensing Amplifiers for Detecting DNA-Interacting Salts and Molecules

Due to the central role of DNA, its interactions with inorganic salts and small organic molecules are important. For example, such interactions play important roles in various fundamental cellular processes in living systems and are involved in many DNA-damage related diseases. Strategies to improve the sensitivity of existing techniques for studying DNA interactions with other molecules would be appreciated in situations where the interactions are too weak. Here we report our development and demonstration of bent DNA bows for amplifying, sensing, and detecting the interactions of 14 inorganic salts and small organic molecules with DNA. With the bent DNA bows, these interactions were easily visualized and quantified in gel electrophoresis, which were difficult to measure without bending. In addition, the strength of the interactions of DNA with the various salts/molecules were quantified using the modified Hill equation. This work highlights the amplification effects of the bending elastic energy stored in the DNA bows and the potential use of the DNA bows for quantitatively measuring DNA interactions with small molecules as simple economic methods; it may also pave the way for exploiting the bent DNA bows for other applications such as screening DNA-interacting molecules and drugs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structure and Interdigitation of Chain-Asymmetric Phosphatidylcholines and Milk Sphingomyelin in the Fluid Phase

We addressed the frequent occurrence of mixed-chain lipids in biological membranes and their impact on membrane structure by studying several chain-asymmetric phosphatidylcholines and the highly asymmetric milk sphingomyelin. Specifically, we report trans-membrane structures of the corresponding fluid lamellar phases using small-angle X-ray and neutron scattering, which were jointly analyzed in terms of a membrane composition-specific model, including a headgroup hydration shell. Focusing on terminal methyl groups at the bilayer center, we found a linear relation between hydrocarbon chain length mismatch and the methyl-overlap for phosphatidylcholines, and a non-negligible impact of the glycerol backbone-tilting, letting the sn1-chain penetrate deeper into the opposing leaflet by half a CH 2 group. That is, penetration-depth differences due to the ester-linked hydrocarbons at the glycerol backbone, previously reported for gel phase structures, also extend to the more relevant physiological fluid phase, but are significantly reduced. Moreover, milk sphingomyelin was found to follow the same linear relationship suggesting a similar tilt of the sphingosine backbone. Complementarily performed molecular dynamics simulations revealed that there is always a part of the lipid tails bending back, even if there is a high interdigitation with the opposing chains. The extent of this back-bending was similar to that in chain symmetric bilayers. For both cases of adaptation to chain length mismatch, chain-asymmetry has a large impact on hydrocarbon chain ordering, inducing disorder in the longer of the two hydrocarbons.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

NCSU RDMA data

NCSU RDMA Data Level b1: QC checks applied to measurements Data Format: CSV Description: See "instrument description Site: Houston, TX; Tracking Aerosol Convection interactions ExpeRiment (HOU) Location: Houston, TX; AMF1 (main site for TRACER) Facility Code: M1 Category: Aerosol Properties Data Type: PI Data Source Instrument/Data: Nano-Scanning Mobility Particle (SMPS); A Radial Differential Mobility Analyzer (RDMA) coupled with 1 Condensation Particle Counter Start Date: 2022-06-01 End Date: 2022-9-26 Contact PI: Markus Petters (mdpetter@ncsu.edu) Funding Source: DOE ASR award US Department of Energy, Office of Science, Biological and Environment Research (grant no. DE-SC 0021074) Instrument description The NCSU RDMA was operated at a sheath-to-sample flow ratio of 5:1.5 L min−1. The RDMA was configured to scan from 5 to 55 nm. It was located into the temperature-controlled trailer adjacent to the NCSU Flux Tower to observe size distributions of the aerosols.The sample line was dried with three silica-gel driers in series, and then neutralized with X-ray neutralizer. Please contact mdpetter@ncsu.edu for further information.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

NCSU HTDMA data

NCSU HTDMA data Data Level b1: QC checks applied to measurements Data Format: CSV Description: See "instrument description Site: Houston, TX; Tracking Aerosol Convection interactions ExpeRiment (HOU) Location: Houston, TX; AMF1 (main site for TRACER) Facility Code: M1 Category: Aerosol Properties Data Type: PI Data Source Instrument/Data: Humidified Tandem Differential Mobility Analyzer; 2 Differential Mobility Analyzers (DMA1 and DMA2). Start Date: 2022-06-01 End Date: 2022-09-26 Contact PI: Markus Petters (mdpetter@ncsu.edu) Funding Source: DOE ASR award US Department of Energy, Office of Science, Biological and Environment Research (grant no. DE-SC 0021074) Instrument description The NCSU HTDMA was operated at a sheath-to-sample flow ratio of 5:1 L min−1. The HTDMA was configured to measure hygroscopic growth factors of dry particles with mobility diameters of D = 15, 20, 30, 40, and 50 nm at RH ~ 70%. A complete cycle for all diameters took ~30 minutes. A sample line brought aerosol inside the trailer at 2.5 L min-1, where it was distributed between NCSU RDMA (1.5 L min-1) and NCSU HTDMA (1 L min-1) lines. The sample line was dried with three silica-gel driers in series, and then neutralized with X-ray neutralizer. The sample line entered DMA1 (operated as an electrostatic classifier). Monodisperse particles with certain fractions were humidified with temperature controlled Nafion membrane immersed in water before entering DMA2 (operated in scanning mobility particle sizer). Please contact mdpetter@ncsu.edu for further information.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Arabidopsis GALACTURONOSYLTRANSFERASE (GAUT) 1 synthesizes a homogalacturonan tightly bound to the cell wall and required for cell expansion

Arabidopsis GALACTURONOSYLTRANSFERASE1 (GAUT1) synthesizes homogalacturonan (HG), the most abundant pectin in growing plant cells. GAUT1 has the greatest in vitro enzyme activity of the six confirmed Arabidopsis HG biosynthetic GAUTs, but its biological activity remains elusive. Here we show that Arabidopsis GAUT1 homozygous mutants have a severe dwarfed seedling phenotype, survive several weeks as 2 to 3 mm seedlings, and have severely reduced shoot and root growth and hypocotyl epidermal, cortex and endodermal cell size. gaut1-1 pollen tubes are shorter than WT with increased bursting. Complementation of homozygous gaut1-1 with GAUT1 coding sequence driven by the GAUT1 promoter restored WT-like growth. The extreme dwarf phenotype of homozygous gaut1-1 seedlings precluded their use for detailed cell wall analysis, thus suspensions cultures were produced from callus generated from mutant and WT seedlings. Homozygous gaut1-1 suspension cells were smaller than WT with ∼30% reduced wall GalA content compared to WT. Sequential extraction of the walls with increasingly harsh solvents and sugar composition analysis revealed reduced GalA content in only the 4M KOH post-chlorite fraction, indicating that GAUT1-synthesized HG was held tightly in the wall by direct or indirect hydrogen bonding and/or oxidation-sensitive linkages. Treatment of wall fractions with endopolygalacturonase to hydrolyze HG and gel electrophoretic separation of hydrolysates exposed an HG-associated doublet band markedly downregulated in the homozygous gaut1-1 4M KOH post-chlorite fraction and to a lesser extent in 4M KOH and sodium chlorite fractions. NMR analysis identified the band as rhamnogalacturonan (RG)-II. Super resolution microscopy using anti-HG antibodies showed that, compared to WT, the homozygous gaut1-1 hypocotyl epidermal and callus cells had reduced content and length of HG nanofilaments, HG fibers associated with cell expansion in Arabidopsis. The results demonstrate that GAUT1-synthesized HG resides in a tightly-cell-wall-bound, RG-II-containing polymer required for HG nanofilament formation and seedling cell expansion.

Atmodjo↗