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At least 757 records · Page 42
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Substrate availability and not thermal acclimation controls microbial temperature sensitivity response to long-term warming

Microbes are responsible for cycling carbon (C) through soils, and predicted changes in soil C stocks under climate change are highly sensitive to shifts in the mechanisms assumed to control the microbial physiological response to warming. Two mechanisms have been suggested to explain the long-term warming impact on microbial physiology: microbial thermal acclimation and changes in the quantity and quality of substrates available for microbial metabolism. Yet studies disentangling these two mechanisms are lacking. To resolve the drivers of changes in microbial physiology in response to long-term warming, we sampled soils from 13- and 28-year-old soil warming experiments in different seasons. We performed short-term laboratory incubations across a range of temperatures to measure the relationships between temperature sensitivity of physiology (growth, respiration, carbon use efficiency, and extracellular enzyme activity) and the chemical composition of soil organic matter. We observed apparent thermal acclimation of microbial respiration, but only in summer, when warming had exacerbated the seasonally-induced, already small dissolved organic matter pools. Irrespective of warming, greater quantity and quality of soil carbon increased the extracellular enzymatic pool and its temperature sensitivity. We propose that fresh litter input into the system seasonally cancels apparent thermal acclimation of C-cycling processes to decadal warming. Our findings reveal that long-term warming has indirectly affected microbial physiology via reduced C availability in this system, implying that earth system models including these negative feedbacks may be best suited to describe long-term warming effects on these soils.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Effects of experimental nitrogen deposition on soil organic carbon storage in Southern California drylands

Atmospheric nitrogen (N) deposition is enriching soils with N across biomes. Soil N enrichment can increase plant productivity and affect microbial activity, thereby increasing soil organic carbon (SOC), but such responses vary across biomes. Drylands cover ~45% of Earth's land area and store ~33% of global SOC contained in the top 1 m of soil. Nitrogen fertilization could, therefore, disproportionately impact carbon (C) cycling, yet whether dryland SOC storage increases with N remains unclear. To understand how N enrichment may change SOC storage, we separated SOC into plant–derived, particulate organic C (POC), and largely microbially derived, mineral–associated organic C (MAOC) at four N deposition experimental sites in Southern California. Theory suggests that N enrichment increases the efficiency by which microbes build MAOC (C stabilization efficiency) if soil pH stays constant. But if soils acidify, a common response to N enrichment, then microbial biomass and enzymatic organic matter decay may decrease, increasing POC but not MAOC. We found that N enrichment had no effect on C fractions except for a decrease in MAOC at one site. Specifically, despite reported increases in plant biomass in three sites and decreases in microbial biomass and extracellular enzyme activities in two sites that acidified, POC did not increase. Furthermore, microbial C use and stabilization efficiency increased in a non–acidified site, but without increasing MAOC. Instead, MAOC decreased by 16% at one of the sites that acidified, likely because it lost 47% of the exchangeable calcium (Ca) relative to controls. Indeed, MAOC was strongly and positively affected by Ca, which directly and, through its positive effect on microbial biomass, explained 58% of variation in MAOC. Finally, long–term effects of N fertilization on dryland SOC storage appear abiotic in nature, such that drylands where Ca–stabilization of SOC is prevalent and soils acidify, are most at risk for significant C loss.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Nitrogen Deposition Weakens Soil Carbon Control of Nitrogen Dynamics Across the Contiguous United States

ABSTRACT Anthropogenic nitrogen (N) deposition is unequally distributed across space and time, with inputs to terrestrial ecosystems impacted by industry regulations and variations in human activity. Soil carbon (C) content normally controls the fraction of mineralized N that is nitrified ( ƒ nitrified ), affecting N bioavailability for plants and microbes. However, it is unknown whether N deposition has modified the relationships among soil C, net N mineralization, and net nitrification. To test whether N deposition alters the relationship between soil C and net N transformations, we collected soils from coniferous and deciduous forests, grasslands, and residential yards in 14 regions across the contiguous United States that vary in N deposition rates. We quantified rates of net nitrification and N mineralization, soil chemistry (soil C, N, and pH), and microbial biomass and function (as beta‐glucosidase (BG) and N ‐acetylglucosaminidase (NAG) activity) across these regions. Following expectations, soil C was a driver of ƒ nitrified across regions, whereby increasing soil C resulted in a decline in net nitrification and ƒ nitrified . The ƒ nitrified value increased with lower microbial enzymatic investment in N acquisition (increasing BG:NAG ratio) and lower active microbial biomass, providing some evidence that heterotrophic microbial N demand controls the ammonium pool for nitrifiers. However, higher total N deposition increased ƒ nitrified , including for high soil C sites predicted to have low ƒ nitrified , which decreased the role of soil C as a predictor of ƒ nitrified . Notably, the drop in contemporary atmospheric N deposition rates during the 2020 COVID‐19 pandemic did not weaken the effect of N deposition on relationships between soil C and ƒ nitrified . Our results suggest that N deposition can disrupt the relationship between soil C and net N transformations, with this change potentially explained by weaker microbial competition for N. Therefore, past N inputs and soil C should be used together to predict N dynamics across terrestrial ecosystems.

Nieland, Matthew A. [Stockbridge School of Agricul↗
DOE OSTI2022

Switchgrass cropping systems affect soil carbon and nitrogen and microbial diversity and activity on marginal lands

Abstract Switchgrass ( Panicum virgatum L.), as a dedicated bioenergy crop, can provide cellulosic feedstock for biofuel production while improving or maintaining soil quality. However, comprehensive evaluations of how switchgrass cultivation and nitrogen (N) management impact soil and plant parameters remain incomplete. We conducted field trials in three years (2016–2018) at six locations in the North Central Great Lakes Region to evaluate the effects of cropping systems (switchgrass, restored prairie, undisturbed control) and N rates (0, 56 kg N ha −1 year −1 ) on biomass yield and soil physicochemical, microbial, and enzymatic parameters. Switchgrass cropping system yielded an aboveground biomass 2.9–3.3 times higher than the other two systems (Jayawardena et al., unpublished data) but our study found that this biomass accumulation did not reduce soil dissolved organic C, total dissolved N (TDN), or bacterial diversity. The annual aboveground biomass removal for bioenergy feedstock, however, reduced soil microbial biomass C (MBC) and microbial biomass N (MBN) and bacterial richness in the second and third years; despite this, continuous monocropping of switchgrass improved soil TDN, inorganic N, bacterial diversity, and shoot biomass in the second and/or third years compared with the first year. N fertilization increased aboveground biomass yield by 1.2 times and significantly increased soil TDN, MBN, and the shoot biomass of switchgrass compared with the unfertilized control. Locations with higher C and N contents and lower C:N ratio had higher aboveground biomass, MBC, MBN, and the activity of BG, CBH, and UREA enzymes; by contrast, locations with higher pH had higher soil TDN and activity of NAG and LAP enzymes. Our research demonstrates that switchgrass cultivation could improve or maintain soil N content and N fertilization can increase plant biomass yield. The comprehensive data also can inform future biogeochemical models to successfully implement switchgrass for bioenergy production.

09 BIOMASS FUELS↗
DOE OSTI2021

Sustained Ability of a Natural Microbial Community to Remove Nitrate from Groundwater

Abstract Microbial‐mediated nitrate removal from groundwater is widely recognized as the predominant mechanism for nitrate attenuation in contaminated aquifers and is largely dependent on the presence of a carbon‐bearing electron donor. The repeated exposure of a natural microbial community to an electron donor can result in the sustained ability of the community to remove nitrate; this phenomenon has been clearly demonstrated at the laboratory scale. However, in situ demonstrations of this ability are lacking. For this study, ethanol (electron donor) was repeatedly injected into a groundwater well (treatment) for six consecutive weeks to establish the sustained ability of a microbial community to remove nitrate. A second well (control) located upgradient was not injected with ethanol during this time. The treatment well demonstrated strong evidence of sustained ability as evident by ethanol, nitrate, and subsequent sulfate removal up to 21, 64, and 68%, respectively, as compared to the conservative tracer (bromide) upon consecutive exposures. Both wells were then monitored for six additional weeks under natural (no injection) conditions. During the final week, ethanol was injected into both treatment and control wells. The treatment well demonstrated sustained ability as evident by ethanol and nitrate removal up to 20 and 21%, respectively, as compared to bromide, whereas the control did not show strong evidence of nitrate removal (5% removal). Surprisingly, the treatment well did not indicate a sustained and selective enrichment of a microbial community. These results suggested that the predominant mechanism(s) of sustained ability likely exist at the enzymatic‐ and/or genetic‐levels. The results of this study demonstrated the in situ ability of a microbial community to remove nitrate can be sustained in the prolonged absence of an electron donor.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

A bifunctional salvage pathway for two distinct S-adenosylmethionine by-products that is widespread in bacteria, including pathogenic Escherichia coli

S-adenosyl-L-methionine (SAM) is a necessary co-substrate for numerous essential enzymatic reactions including protein and nucleotide methylations, secondary metabolite synthesis, and radical-mediated processes. Radical SAM enzymes produce 5’-deoxyadenosine, and SAM-dependent enzymes for polyamine, neurotransmitter, and quorum sensing compound synthesis produce 5’-methylthioadenosine as byproducts. Both are inhibitory and must be addressed by all cells. This work establishes a bifunctional oxygen-independent salvage pathway for 5’-deoxyadenosine and 5’-methylthioadenosine in both Rhodospirillum rubrum and Extraintestinal Pathogenic Escherichia coli. Homologous genes for this pathway are widespread in bacteria, notably pathogenic strains within several families. A phosphorylase (Rhodospirillum rubrum) or separate nucleoside and kinase (Escherichia coli) followed by an isomerase and aldolase sequentially function to salvage these two wasteful and inhibitory compounds into adenine, dihydroxyacetone phosphate and acetaldehyde or (2-methylthio)acetaldehyde during both aerobic and anaerobic growth. Both SAM byproducts are metabolized with equal affinity during aerobic and anaerobic growth conditions, suggesting that the dual-purpose salvage pathway plays a central role in numerous environments, notably the human body during infection. Finally, our newly discovered bifunctional oxygen-independent pathway, widespread in bacteria, salvages at least two byproducts of SAM-dependent enzymes for carbon and sulfur salvage, contributing to cell growth.

09 BIOMASS FUELS↗
DOE OSTI2022

A new approach to zip–lignin: 3,4–dihydroxybenzoate is compatible with lignification

Renewed interests in the development of bioenergy, biochemicals, and biomaterials have elicited new strategies for engineering the lignin of biomass feedstock plants. Furthermore, this study shows, for the first time, that 3,4-dihydroxybenzoate (DHB) is compatible with the radical coupling reactions that assemble polymeric lignin in plants. We introduced a bacterial 3-dehydroshikimate dehydratase into hybrid poplar (Populus alba × grandidentata) to divert carbon flux away from the shikimate pathway, which lies upstream of lignin biosynthesis. Transgenic poplar wood had up to 33% less lignin with p-hydroxyphenyl units comprising as much as 10% of the lignin. Mild alkaline hydrolysis of transgenic wood released fewer ester-linked p-hydroxybenzoate groups than control trees, and revealed the novel incorporation of cell-wall-bound DHB, as well as glycosides of 3,4-dihydroxybenzoic acid (DHBA). Two-dimensional nuclear magnetic resonance (2D-NMR) analysis uncovered DHBA-derived benzodioxane structures suggesting that DHB moieties were integrated into the lignin polymer backbone. In addition, up to 40% more glucose was released from transgenic wood following ionic liquid pretreatment and enzymatic hydrolysis. This work highlights the potential of diverting carbon flux from the shikimate pathway for lignin engineering and describes a new type of ‘zip-lignin’ derived from the incorporation of DHB into poplar lignin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Stacking AsFMT overexpression with BdPMT loss of function enhances monolignol ferulate production in Brachypodium distachyon

To what degree can the lignin subunits in a monocot be derived from monolignol ferulate (ML-FA) conjugates? This simple question comes with a complex set of variables. Three potential requirements for optimizing ML-FA production are as follows: (1) The presence of an active FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme throughout monolignol production; (2) Suppression or elimination of enzymatic pathways competing for monolignols and intermediates during lignin biosynthesis; and (3) Exclusion of alternative phenolic compounds that participate in lignification. A 16-fold increase in lignin-bound ML-FA incorporation was observed by introducing an AsFMT gene into Brachypodium distachyon. On its own, knocking out the native p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (BdPMT) pathway that competes for monolignols and the p-coumaroyl-CoA intermediate did not change ML-FA incorporation, nor did partial loss of CINNAMOYL-CoA REDUCTASE1 (CCR1) function, which reduced metabolic flux to monolignols. However, stacking AsFMT into the Bdpmt-1 mutant resulted in a 32-fold increase in ML-FA incorporation into lignin over the wild-type level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Towards chloroplastic nanofactories: formation of proteinaceous scaffolds for metabolic engineering

The evolution of eukaryotic lipid-bound organelles allows for specialized metabolism to occur within spatially distinct metabolic landscapes within the same cell. However, this strategy of compartmentalization is not unique to eukaryotic organisms. Many bacteria, spread across 45 different phyla, contain loci that encode for specialized bacterial microcompartments (BMCs) (Sutter et al., 2021). The formation of a BMC involves self-assembly from three families of shell proteins to form the outer shell membrane, in addition to the encapsulation of an enzymatic core packaged within the lumen of a BMC (Kerfeld et al., 2018). These proteinaceous organelles provide a competitive growth advantage by enabling organisms to process inaccessible substrates by sequestering metabolic intermediates (i.e. aldehydes) that would otherwise be toxic within the cytoplasm. While BMCs perform specific metabolic functions in their native organism, synthetic, empty BMC shells can form without the requirement of native cargo inside (Doron and Kerfeld, 2024). This provides a transferable and tunable platform of protein scaffolding for guiding metabolic engineering within a host organism of choice (Raba and Kerfeld, 2022).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Simultaneous Overexpression of FERULOYL‐CoA 6′‐HYDROXYLASE 1 and COUMARIN SYNTHASE Leads to Coumarin‐Enriched Lignin and Improved Saccharification in Greenhouse‐ and Field‐Grown Poplar

ABSTRACT The urgent need for renewable resources has increased the interest in woody biomass to manufacture bio‐based products. However, lignin recalcitrance limits the enzymatic conversion of wood into fermentable sugars, posing a major challenge for biomass deconstruction. To address this problem, we aimed at incorporating the coumarin scopoletin into the lignin polymer of poplar ( Populus tremula × P . alba ) by expressing FERULOYL‐CoA 6′‐HYDROXYLASE 1 ( F6′H1 ) and COUMARIN SYNTHASE ( COSY ) in lignifying cells. Three constructs were evaluated: two bicistronic constructs, SCOP1 ( COSY followed by F6′H1 ) and SCOP2 ( F6′H1 followed by COSY ), and one monocistronic, SCOP3 (only F6′H1 ). SCOP1 poplars produced most free scopoletin without altering overall lignin, cellulose or hemicellulose content. SCOP2 poplars were overall less efficient in scopoletin production and most of these lines showed a severe biomass yield penalty, whereas SCOP3 caused plant lethality. NMR and metabolic analyses confirmed that scopoletin cross‐coupled with G and S monomers during lignification in SCOP1 lines. In addition to scopoletin, the detection of benzodioxane structures revealed the incorporation of dihydroxycoumarins. Overall coumarin incorporation in lignin amounted up to 2.3%. After alkaline pretreatment, wood from greenhouse‐grown SCOP1 poplars released up to 29% more glucose compared to the wild type upon limited saccharification. Field‐testing of three SCOP1 lines showed a 6 to 11% increase in saccharification efficiency, with the line containing the lowest scopoletin levels maintaining normal growth. These results demonstrate that engineering lignin composition in poplar can improve saccharification, and emphasize the importance of construct design, translational research and field validation.

alternative lignin monomers↗
DOE OSTI2022

Cell wall ester modifications and volatile emission signatures of plant response to abiotic stress

Abstract Growth suppression and defence signalling are simultaneous strategies that plants invoke to respond to abiotic stress. Here, we show that the drought stress response of poplar trees ( Populus trichocarpa ) is initiated by a suppression in cell wall derived methanol (MeOH) emissions and activation of acetic acid (AA) fermentation defences. Temperature sensitive emissions dominated by MeOH (AA/MeOH <30%) were observed from physiologically active leaves, branches, detached stems, leaf cell wall isolations and whole ecosystems. In contrast, drought treatment resulted in a suppression of MeOH emissions and strong enhancement in AA emissions together with volatiles acetaldehyde, ethanol, and acetone. These drought‐induced changes coincided with a reduction in stomatal conductance, photosynthesis, transpiration, and leaf water potential. The strong enhancement in AA/MeOH emission ratios during drought (400%–3500%) was associated with an increase in acetate content of whole leaf cell walls, which became significantly 13 C 2 ‐labelled following the delivery of 13 C 2 ‐acetate via the transpiration stream. The results are consistent with both enzymatic and nonenzymatic MeOH and AA production at high temperature in hydrated tissues associated with accelerated primary cell wall growth processes, which are downregulated during drought. While the metabolic source(s) require further investigation, the observations are consistent with drought‐induced activation of aerobic fermentation driving high rates of foliar AA emissions and enhancements in leaf cell wall O ‐acetylation. We suggest that atmospheric AA/MeOH emission ratios could be useful as a highly sensitive signal in studies investigating environmental and biological factors influencing growth‐defence trade‐offs in plants and ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Increased activity of core photorespiratory enzymes and CO 2 transfer conductances are associated with higher and more optimal photosynthetic rates under elevated temperatures in the extremophile Rhazya stricta

Increase photorespiration and optimising intrinsic water use efficiency are unique challenges to photosynthetic carbon fixation at elevated temperatures. To determine how plants can adapt to facilitate high rates of photorespiration at elevated temperatures while also maintaining water-use efficiency, we performed in-depth gas exchange and biochemical assays of the C 3 extremophile, Rhazya stricta. These results demonstrate that R. stricta supports higher rates of photorespiration under elevated temperatures and that these higher rates of photorespiration correlate with increased activity of key photorespiratory enzymes; phosphoglycolate phosphatase and catalase. The increased photorespiratory enzyme activities may increase the overall capacity of photorespiration by reducing enzymatic bottlenecks and allowing minimal inhibitor accumulation under high photorespiratory rates. Additionally, we found the CO 2 transfer conductances (stomatal and mesophyll) are re-allocated to increase the water-use efficiency in R. stricta but not necessarily the photosynthetic response to temperature. These results suggest important adaptive strategies in R. stricta that maintain photosynthetic rates under elevated temperatures with optimal water loss. The strategies found in R. stricta may inform breeding and engineering efforts in other C 3 species to improve photosynthetic efficiency at high temperatures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Rewired phenolic metabolism and improved saccharification efficiency of a Zea mays cinnamyl alcohol dehydrogenase 2 (zmcad2) mutant

Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification, and is a promising target to improve the cellulose–to–glucose conversion of maize stover. Here, we analyzed a field–grown zmcad2 Mutator transposon insertional mutant. Zmcad2 mutant plants had an 18% lower Klason lignin content, whereas their cellulose content was similar to that of control lines. The lignin in zmcad2 mutants contained increased levels of hydroxycinnamaldehydes, i.e. the substrates of ZmCAD2, ferulic acid and tricin. Ferulates decorating hemicelluloses were not altered. Phenolic profiling further revealed that hydroxycinnamaldehydes are partly converted into (dihydro)ferulic acid and sinapic acid and their derivatives in zmcad2 mutants. Syringyl lactic acid hexoside, a metabolic sink in CAD–deficient dicot trees, appeared not to be a sink in zmcad2 maize. The enzymatic cellulose–to–glucose conversion efficiency was determined after 10 different thermochemical pre–treatments. Zmcad2 yielded significantly higher conversions compared with controls for almost every pre–treatment. However, the relative increase in glucose yields after alkaline pre–treatment was not higher than the relative increase when no pre–treatment was applied, suggesting that the positive effect of the incorporation of hydroxycinnamaldehydes was leveled off by the negative effect of reduced p–coumarate levels in the cell wall. Here, taken together, our results reveal how phenolic metabolism is affected in CAD–deficient maize, and further support mutating CAD genes in cereal crops as a promising strategy to improve lignocellulosic biomass for sugar–platform biorefineries.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Rerouting of the lignin biosynthetic pathway by inhibition of cytosolic shikimate recycling in transgenic hybrid aspen

Lignin is a phenolic polymer deposited in the plant cell wall and is mainly polymerized from three canonical monomers (monolignols), i.e., p-coumaryl, coniferyl, and sinapyl alcohols. After polymerization, these alcohols form different lignin substructures. In dicotyledons, monolignols are biosynthesized from phenylalanine, an aromatic amino acid. Shikimate acts at two positions in the route to the lignin building blocks. It is part of the shikimate pathway which provides the precursor for the biosynthesis of phenylalanine, and is involved in the transesterification of pcoumaroyl- CoA to p-coumaroyl shikimate, one of key steps in the biosynthesis of coniferyl and sinapyl alcohols. The shikimate residue in p-coumaroyl shikimate is released in later steps, and the resulting shikimate becomes available again for the biosynthesis of new p-coumaroyl shikimate molecules. In this study, we inhibited cytosolic shikimate recycling in transgenic hybrid aspen by accelerated phosphorylation of shikimate in the cytosol through expression of a bacterial shikimate kinase. Here, this expression elicited an increase in p-hydroxyphenyl units of lignin and, by contrast, a decrease in guaiacyl and syringyl units. Transgenic plants with high shikimate kinase activity produced a lignin content comparable to that in wild-type plants and had an increased processability via enzymatic saccharification. Although expression of many genes was altered in the transgenic plants, elevated shikimate kinase activity did not exert a significant effect on the expression of the majority of genes responsible for lignin biosynthesis. The present results indicate that cytosolic shikimate recycling is crucial to the monomeric composition of lignin rather than for lignin content.

shikimate pathway↗
DOE OSTI2023

Plant terpene specialized metabolism: complex networks or simple linear pathways?

From the perspectives of pathway evolution, discovery and engineering of plant specialized metabolism, the nature of the biosynthetic routes represents a critical aspect. Classical models depict biosynthesis typically from an end-point angle and as linear, for example, connecting central and specialized metabolism. As the number of functionally elucidated routes increased, the enzymatic foundation of complex plant chemistries became increasingly well understood. The perception of linear pathway models has been severely challenged. With a focus on plant terpenoid specialized metabolism, we review here illustrative examples supporting that plants have evolved complex networks driving chemical diversification. The completion of several diterpene, sesquiterpene and monoterpene routes shows complex formation of scaffolds and their subsequent functionalization. These networks show that branch points, including multiple sub-routes, mean that metabolic grids are the rule rather than the exception. This concept presents significant implications for biotechnological production.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Arabidopsis stromal carbonic anhydrases exhibit non–overlapping roles in photosynthetic efficiency and development

Carbonic anhydrases (CAs) are ubiquitous enzymes that accelerate the reversible conversion of CO 2 to HCO 3 – . The Arabidopsis genome encodes members of the α-, β- and γ-CA families, and it has been hypothesized that βCA activity has a role in photosynthesis. In this work, we tested this hypothesis by characterizing the two plastidial βCAs, βCA1 and βCA5, in physiological conditions of growth. We conclusively established that both proteins are localized in the chloroplast stroma and that the loss of βCA5 induced the expression of βCA1, supporting the existence of regulatory mechanisms to control the expression of stromal βCAs. We also established that βCA1 and βCA5 have markedly different enzymatic kinetics and physiological relevance. Specifically, we found that βCA5 had a first-order rate constant ~10-fold lower than βCA1, and that the loss of βCA5 is detrimental to growth and could be rescued by high CO 2 . Furthermore, we established that, while a βCA1 mutation showed near wild-type growth and no significant impact on photosynthetic efficiency, the loss of βCA5 markedly disrupted photosynthetic efficiency and light-harvesting capacity at ambient CO 2 . Therefore, we conclude that in physiological autotrophic growth, the loss of the more highly expressed βCA1 does not compensate for the loss of a less active βCA5, which in turn is involved in growth and photosynthesis at ambient CO 2 levels. These results lend support to the hypothesis that, in Arabidopsis,βCAs have non-overlapping roles in photosynthesis and identify a critical activity of stromal βCA5 and a dispensable role for βCA1.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Identification of glycosyltransferases mediating 2‐ O ‐arabinopyranosyl and 2‐ O ‐galactosyl substitutions of glucuronosyl side chains of xylan

SUMMARY Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O ‐2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2‐ O ‐arabinopyranose (Ara p ) or 2‐ O ‐galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3‐clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Ara p from UDP‐Ara p onto O ‐2 of GlcA side chains of xylan, and thus it was named xylan 2‐ O ‐arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T‐DNA knockout mutation showing a loss of the Ara p substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus , lemon‐scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2‐ O ‐Ara p and 2‐ O ‐Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site‐directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2‐ O ‐arabinopyranosylation and 2‐ O ‐galactosylation of GlcA side chains of xylan.

Zhong, Ruiqin↗
DOE OSTI2020

2.7 Å cryo-EM structure of rotavirus core protein VP3, a unique capping machine with a helicase activity

In many viruses, including rotavirus (RV), the major pathogen of infantile gastroenteritis, capping of viral messenger RNAs is a pivotal step for efficient translation of the viral genome. In RV, VP3 caps the nascent transcripts synthesized from the genomic dsRNA segments by the RV polymerase VP1 within the particle core. Here, from cryo–electron microscopy, x-ray crystallography, and biochemical analyses, we show that VP3 forms a stable tetrameric assembly with each subunit having a modular domain organization, which uniquely integrates five distinct enzymatic steps required for capping the transcripts. In addition to the previously known guanylyl- and methyltransferase activities, we show that VP3 exhibits hitherto unsuspected RNA triphosphatase activity necessary for initiating transcript capping and RNA helicase activity likely required for separating the RNA duplex formed transiently during endogenous transcription. From our studies, we propose a new mechanism for how VP3 inside the virion core caps the nascent transcripts exiting from the polymerase.

59 BASIC BIOLOGICAL SCIENCES↗