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At least 325 records · Page 18
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

SEED LIPID DROPLET PROTEIN1, SEED LIPID DROPLET PROTEIN2, and LIPID DROPLET PLASMA MEMBRANE ADAPTOR mediate lipid droplet–plasma membrane tethering

Membrane contact sites (MCSs) are interorganellar connections that allow for the direct exchange of molecules, such as lipids or Ca 2+ between organelles, but can also serve to tether organelles at specific locations within cells. Here, we identified and characterized three proteins of Arabidopsis thaliana that form a lipid droplet (LD)–plasma membrane (PM) tethering complex in plant cells, namely LD-localized SEED LD PROTEIN (SLDP) 1 and SLDP2 and PM-localized LD-PLASMA MEMBRANE ADAPTOR (LIPA). Using proteomics and different protein–protein interaction assays, we show that both SLDPs associate with LIPA. Disruption of either SLDP1 and SLDP2 expression, or that of LIPA, leads to an aberrant clustering of LDs in Arabidopsis seedlings. Ectopic co-expression of one of the SLDPs with LIPA is sufficient to reconstitute LD–PM tethering in Nicotiana tabacum pollen tubes, a cell type characterized by dynamically moving LDs in the cytosolic streaming. Furthermore, confocal laser scanning microscopy revealed both SLDP2.1 and LIPA to be enriched at LD–PM contact sites in seedlings. These and other results suggest that SLDP and LIPA interact to form a tethering complex that anchors a subset of LDs to the PM during post-germinative seedling growth in Arabidopsis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

GTP binding by Arabidopsis extra-large G protein 2 is not essential for its functions

The extra-large guanosine-5'-triphosphate (GTP)-binding protein 2, XLG2, is an unconventional Gα subunit of the Arabidopsis (Arabidopsis thaliana) heterotrimeric GTP-binding protein complex with a major role in plant defense. In vitro biochemical analyses and molecular dynamic simulations show that affinity of XLG2 for GTP is two orders of magnitude lower than that of the conventional Gα, AtGPA1. Here we tested the physiological relevance of GTP binding by XLG2. We generated an XLG2(T476N) variant with abolished GTP binding, as confirmed by in vitro GTPγS binding assay. Yeast three-hybrid, bimolecular fluorescence complementation, and split firefly-luciferase complementation assays revealed that the nucleotide-depleted XLG2(T476N) retained wild-type XLG2-like interactions with the Gβγ dimer and defense-related receptor-like kinases. Both wild-type and nucleotide-depleted XLG2(T476N) restored the defense responses against Fusarium oxysporum and Pseudomonas syringae compromised in the xlg2 xlg3 double mutant. Additionally, XLG2(T476N) was fully functional restoring stomatal density, root growth, and sensitivity to NaCl, but failed to complement impaired germination and vernalization-induced flowering. We conclude that XLG2 is able to function in a GTP-independent manner and discuss its possible mechanisms of action.

Plant Sciences↗
DOE OSTI2024

Propelling sustainable energy: Multi-omics analysis of pennycress FATTY ACID ELONGATION1 knockout for biofuel production

Abstract The aviation industry’s growing interest in renewable jet fuel has encouraged the exploration of alternative oilseed crops. Replacing traditional fossil fuels with a sustainable, domestically sourced crop can substantially reduce carbon emissions, thus mitigating global climate instability. Pennycress (Thlaspi arvense L.) is an emerging oilseed intermediate crop that can be grown during the offseason between maize (Zea mays) and soybean (Glycine max) to produce renewable biofuel. Pennycress is being domesticated through breeding and mutagenesis, providing opportunities for trait enhancement. Here, we employed metabolic engineering strategies to improve seed oil composition and bolster the plant's economic competitiveness. FATTY ACID ELONGATION1 (FAE1) was targeted using CRISPR-Cas 9 gene editing to eliminate very long chain fatty acids (VLCFAs) from pennycress seed oil, thereby enhancing its cold flow properties. Through an integrated multiomics approach, we investigated the impact of eliminating VLCFAs in developing and mature plant embryos. Our findings revealed improved cold-germination efficiency in fae1, with seedling emergence occurring up to 3 d earlier at 10 °C. However, these alterations led to a tradeoff between storage oil content and composition. Additionally, these shifts in lipid biosynthesis were accompanied by broad metabolic changes, such as the accumulation of glucose and ADP-glucose quantities consistent with increased starch production. Furthermore, shifts to shorter FA chains triggered the upregulation of heat shock proteins, underscoring the importance of VLCFAs in stress signaling pathways. Overall, this research provides crucial insights for optimizing pennycress seed oil while preserving essential traits for biofuel applications.

Rasoul, Amira (ORCID:0009000714290175)↗
DOE OSTI2019

Microscopic, Biochemical, and Molecular Comparisons of Moderately Resistant and Susceptible Populus Genotypes Inoculated with Sphaerulina musiva

Sphaerulina musiva, the causal agent of Septoria leaf spot and stem canker, is responsible for mortality and yield loss in Populus plantations. However, little is known about the mode of infection and the mechanisms of resistance in this pathosystem. To characterize these phenomena, microscopic, biochemical, and transcriptome comparisons were performed between leaves of moderately resistant and susceptible genotypes of Populus inoculated with S. musiva conidia. Using scanning electron, cryofracture, and laser-scanning confocal microscopy, the infection and colonization of Populus leaves by S. musiva were examined across five time points (48 h, 96 h, 1 week, 2 weeks, and 3 weeks). The infection process was similar regardless of the host genotype. Differences in host colonization between susceptible and moderately resistant genotypes were apparent by 1 week postinoculation. However, the germination of conidia was greater on the susceptible than on the moderately resistant genotype (P < 0.008). Diaminobenzidine staining, a measure of hydrogen peroxide accumulation, was different (P < 0.001) between the host genotypes by 2 weeks postinoculation. Transcriptome differences between genotypes indicated that the speed and amplitude of the defense response were faster and more extensive in the moderately resistant genotype. Changes in gene expression support the microscopic and biochemical observations.

Plant Sciences↗
DOE OSTI2016

Chemical Biology And Endomembrane Trafficking In Plants

Endomembrane trafficking is essential for coordinated growth and development in plants and response to environment. In particular, key plasma membrane proteins such as PIN auxin transporters and the BRI1 brassinosteroid receptor translocate dynamically between the endosomes and plasma membrane. We utilized a previously published large‐scale chemical library screening approach in which we identified small molecules that inhibited pollen germination or tube growth. This tip growth requires exocytosis, endocytosis and other endomembrane trafficking processes We then identified groups of small molecules that affect trafficking of PIN auxin transporters and other plasma membrane proteins in Arabidopsis roots. One of these compounds (ES2) targets the essential EXO70A1 component of the exocyst complex in Arabidopsis which is necessary for normal development. In Arabidopsis, there are 23 EXO70 genes (in mammals and yeast they are low copy number) where, among other roles, they appear to be involved in autophagy, cell division and pathogen responses. ES2 targets the exocyst complex in plants as well as mammals including humans where defective recycling/exocytosis have been correlated with disease. To our knowledge, ES2 is the first bioactive compound known to have this specificity. To examine exocytosis and its regulation in more detail, we have examined exo70 mutants, generated EXO70A1 structural data and are examining additional small molecules that may affect recycling/exocytosis. Support or Funding Information Acknowledgement: This work was supported by the US Department of Energy DE‐FG02‐02ER15295 (GRH, NVR).

Hicks, Glenn R.↗
DOE OSTI2021

Analysis of putative quadruplex-forming sequences in fungal genomes: novel antifungal targets?

Fungal infections cause >1 million deaths annually and the emergence of antifungal resistance has prompted the exploration for novel antifungal targets. Quadruplexes are four-stranded nucleic acid secondary structures, which can regulate processes such as transcription, translation, replication and recombination. They are also found in genes linked to virulence in microbes, and ligands that bind to quadruplexes can eliminate drug-resistant pathogens. Using a computational approach, we quantified putative quadruplex-forming sequences (PQS) in 1359 genomes across the fungal kingdom and explored their presence in genes related to virulence, drug resistance and biological processes associated with pathogenicity in Aspergillus fumigatus. Here we present the largest analysis of PQS in fungi and identify significant heterogeneity of these sequences throughout phyla, genera and species. PQS were genetically conserved in Aspergillus spp. and frequently pathogenic species appeared to contain fewer PQS than their lesser/non-pathogenic counterparts. GO-term analysis identified that PQS-containing genes were involved in processes linked with virulence such as zinc ion binding, the biosynthesis of secondary metabolites and regulation of transcription in A. fumigatus. Although the genome frequency of PQS was lower in A. fumigatus, PQS could be found enriched in genes involved in virulence, and genes upregulated during germination and hypoxia. Moreover, PQS were found in genes involved in drug resistance. Quadruplexes could have important roles within fungal biology and virulence, but their roles require further elucidation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Systematic Phenotypic Screen of Arabidopsis Peroxisomal Mutants Identifies Proteins Involved in β -Oxidation

Peroxisomes are highly dynamic and multifunctional organelles essential to development. Plant peroxisomes accommodate a multitude of metabolic reactions, many of which are related to the β-oxidation of fatty acids or fatty acid-related metabolites. Recently, several dozens of novel peroxisomal proteins have been identified from Arabidopsis (Arabidopsis thaliana) through in silico and experimental proteomic analyses followed by in vivo protein targeting validations. To determine the functions of these proteins, we interrogated their transfer DNA insertion mutants with a series of physiological, cytological, and biochemical assays to reveal peroxisomal deficiencies. Sugar dependence and 2,4-dichlorophenoxybutyric acid and 12-oxo-phytodienoic acid response assays uncovered statistically significant phenotypes in β-oxidation-related processes in mutants for 20 of 27 genes tested. Additional investigations uncovered a subset of these mutants with abnormal seed germination, accumulation of oil bodies, and delayed degradation of long-chain fatty acids during early seedling development. Mutants for seven genes exhibited deficiencies in multiple assays, strongly suggesting the involvement of their gene products in peroxisomal β-oxidation and initial seedling growth. Proteins identified included isoforms of enzymes related to β-oxidation, such as acyl-CoA thioesterase2, acyl-activating enzyme isoform1, and acyl-activating enzyme isoform5, and proteins with functions previously unknown to be associated with b-oxidation, such as Indigoidine synthase A, Senescence-associated protein/B12D-related protein1, Betaine aldehyde dehydrogenase, and Unknown protein5. This multipronged phenotypic screen allowed us to reveal b-oxidation proteins that have not been discovered by single assay-based mutant screens and enabled the functional dissection of different isoforms of multigene families involved in β-oxidation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. Furthemore, these analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. Our analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Growth strategies and threshold responses to water deficit modulate effects of warming on tree seedlings from forest to alpine

Predictions of upslope range shifts for tree species with warming are based on assumptions of moisture stress at lower elevation limits and low-temperature stress at high-elevation limits. However, recent studies have shown that warming can reduce tree seedling establishment across the entire gradient from subalpine forest to alpine via moisture limitation. Warming effects also vary with species, potentially resulting in community shifts in high-elevation forests. We examined the growth and physiology underlying effects of warming on seedling demographic patterns. We evaluated dry mass (DM), root length, allocation above- and below-ground, and relative growth rate (RGR) of whole seedlings, and their ability to avoid or endure water stress via water-use efficiency and resisting turgor loss, for Pinus flexilis , Picea engelmannii and Pinus contorta seeded below, at and above tree line in experimentally warmed, watered and control plots in the Rocky Mountains, USA. We expected that growth and allocation responses to warming would relate to moisture status and that variation in drought tolerance traits would explain species differences in survival rates. Across treatments and elevations, seedlings of all species had weak turgor-loss resistance, and growth was marginal with negative RGR in the first growth phase (-0.01 to -0.04 g g -1 day -1 ). Growth was correlated with soil moisture, particularly in the relatively small-seeded P. contorta and P. engelmannii . Pinus flexilis , known to have the highest survivorship, attained the greatest DM and longest root but was also the slowest growing and most water-use efficient. This was likely due to its greater reliance on seed reserves. Seedlings developed 15% less total DM, 25% less root DM and 11% shorter roots in heated compared with unheated plots. Higher temperatures slightly increased DM, root length and RGR where soils were wettest, but more strongly decreased these variables under drier conditions. Synthesis. The surprising heat inhibition of tree seedling establishment at the cold edge of forests appears to have a physiological basis: newly germinated seedlings have poor moisture stress tolerance, which appears related to marginal initial growth and heavy reliance on seed reserves. Variation in these attributes among tree species at tree line helps explain their different climate responses.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2018

A century of genetic variation inferred from a persistent soil‐stored seed bank

Abstract Stratigraphic accretion of dormant propagules in soil can result in natural archives useful for studying ecological and evolutionary responses to environmental change. Few attempts have been made, however, to use soil‐stored seed banks as natural archives, in part because of concerns over nonrandom attrition and mixed stratification. Here, we examine the persistent seed bank of Schoenoplectus americanus , a foundational brackish marsh sedge, to determine whether it can serve as a resource for reconstructing historical records of demographic and population genetic variation. After assembling profiles of the seed bank from radionuclide‐dated soil cores, we germinated seeds to “resurrect” cohorts spanning the 20th century. Using microsatellite markers, we assessed genetic diversity and differentiation among depth cohorts, drawing comparisons to extant plants at the study site and in nearby and more distant marshes. We found that seed density peaked at intermediate soil depths. We also detected genotypic differences among cohorts as well as between cohorts and extant plants. Genetic diversity did not decline with depth, indicating that the observed pattern of differentiation is not due to attrition. Patterns of differentiation within and among extant marshes also suggest that local populations persist as aggregates of small clones, likely reflecting repeated seedling recruitment and low immigration from admixed regional gene pools. These findings indicate that persistent and stratified soil‐stored seed banks merit further consideration as resources for reconstructing decadal‐ to century‐long records that can lend insight into the tempo and nature of ecological and evolutionary processes that shape populations over time.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2019

Cryptic phenology in plants: case studies, implications and recommendations

Plant phenology – the timing of cyclic or recurrent biological events in plants – offers insight into the ecology, evolution, and seasonality of plant–mediated ecosystem processes. Traditionally studied phenologies are readily apparent, such as flowering events, germination timing, and season–initiating budbreak. However, a broad range of phenologies that are fundamental to the ecology and evolution of plants, and to global biogeochemical cycles and climate change predictions, have been neglected because they are “cryptic” – that is, hidden from view (e.g root production) or difficult to distinguish and interpret based on common measurements at typical scales of examination (e.g leaf turnover in evergreen forests). We illustrate how capturing cryptic phenology can advance scientific understanding with two case studies: wood phenology in a deciduous forest of the northeastern USA and leaf phenology in tropical evergreen forests of Amazonia. Drawing on these case studies and other literature, we argue that conceptualizing and characterizing cryptic plant phenology is needed for understanding and accurate prediction at many scales from organisms to ecosystems. As a result, we recommend avenues of empirical and modeling research to accelerate discovery of cryptic phenological patterns, to understand their causes and consequences, and to represent these processes in terrestrial biosphere models.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

An efficient virus–induced gene silencing (VIGS) system for gene functional studies in Miscanthus

Virus-induced gene silencing (VIGS) is a powerful tool for transient gene functional analysis in plants, especially for monocot species (e.g., grasses) that are recalcitrant to transformation. Despite various VIGS systems that have been developed in different plant species, none was previously available for the bioenergy crop Miscanthus. Here, we report the establishment of an efficient and robust VIGS system mediated by Tobacco Rattle Virus (TRV) in Miscanthus. We first investigated the impact of various factors that may affect gene silencing efficiency using the Miscanthus sinensis Phytoene Desaturase (MsPDS) gene as a visual indicator of photobleaching. Then, we optimized the TRV-elicited VIGS procedure using an orthogonal experimental design with four factors (sprout size, Agrobacterium concentration, vacuum infiltration time, and co-incubation time) each at three levels. The following led to the highest silencing efficiency (~76%): inoculation of germinating seedlings (1.0–2.0 mm), Agrobacterium tumefaciens culture grown to optical density at 600 nm (OD 600 ) of 0.4, vacuum infiltration for 90 min, and co-incubation for 5 h. The VIGS system established was applicable for both M. sinensis and M. lutarioriparius, with comparable gene silencing efficiency. We verified the efficacy of the VIGS system via the functional characterization of the role of a MYB transcription factor, MsMYB112, in salt stress tolerance. Expression of MsMYB112 was successfully knocked down using the VIGS system, and this led to compromised salt tolerance in the silenced Miscanthus plants. The TRV-based VIGS system established may, therefore, substantially facilitate functional genomic studies in Miscanthus.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Biomass yields, reproductive fertility, compositional analysis, and genetic diversity of newly developed triploid giant miscanthus hybrids

Abstract Miscanthus × giganteus (giant miscanthus), first found as a naturally occurring hybrid, has shown promise as a bioenergy/biomass crop throughout much of the temperate world. This allotriploid (2 n = 3 x = 57) hybrid resulted from a cross between tetraploid Miscanthus sacchariflorus (2 n = 4 x = 76) and diploid Miscanthus sinensis (2 n = 2 x = 38) and is particularly desirable due to its low fertility that minimizes reseeding and potential invasiveness. However, there is limited genetic diversity in commonly grown cultivars of triploid M. × giganteus and breeding and development efforts to improve and domesticate this crop have been minimal. Here, we report on newly developed M. × giganteus hybrids compared with the industry standard M. × giganteus '1993‐1780'. Dry biomass yields of new hybrids ranged from 19.5 to 32.4 Mg/ha/year for the fourth growing season, compared with 21.0 Mg/ha/year for M. × giganteus '1993‐1780'. Plant reproductive fertility remained low for all accessions with overall fertility [(seed set × seed germination)/100] ranging from 0.3% to 4.5% for new hybrids compared to 0.4% for M. × giganteus '1993‐1780'. Culm density and height varied among accessions and were positively correlated with increased biomass. Based on compositional analyses, theoretical ethanol yields ranged from 9, 740 to 16,278 L/ha/year for new hybrids compared to 10,406 L/ha/year for M. × giganteus '1993‐1780'. Relative feed value indices were low overall and ranged between 66.0 and 72.8 for new hybrids compared to M. × giganteus '1993‐1780' with 71.3. The genetic diversity of new hybrids, compared with existing cultivars, was characterized using whole genome sequences. Based on pair‐wise distances, cluster analysis clearly showed increased diversity of new hybrids compared with earlier selections. These results document new triploid hybrids of M. × giganteus with enhanced biomass and theoretical ethanol yields in combination with broader genetic diversity and lowreproductive fertility.

Touchell, Darren H.↗
DOE OSTI2021

All in the timing: epigenetic control of greening

During germination, the emerging seedling relies on a limited supply of energy and nutrients stored within itself and the seed. This often occurs underground in darkness where seedlings undergo skotomorphogenesis, characterized by hypocotyl elongation, reduced root growth, and small closed cotyledons forming an apical hook to protect the shoot apical meristem as the seedling grows though the soil towards sunlight. Such long and pale dark-grown seedlings are commonly referred to as etiolated (from the French étiolier (to make pale)). However, a seedling must eventually find sunlight and make an irreversible shift to photoautotrophic growth (photomorphogenesis or de-etiolation). This leads to a dramatic change in physiology, including reduced hypocotyl elongation, increased root growth, expansion and unfolding of the cotyledons, and the biogenesis of photosynthetic plastids (i.e. chloroplasts). Furthermore, this shift is accompanied by a massive change in gene expression, with about one-third of genes being reprogrammed. A large amount of the transcriptional response is regulated by the plant photoreceptors (phytochromes and cryptochromes) that activate transcriptional networks. Furthermore, this involves the rapid degradation of PHYTOCHROME INTERACTING FACTORS (PIFs), which are basic helix-loop-helix domain-containing transcription factors that repress photomorphogenesis in the dark.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Forest regeneration within Earth system models: current process representations and ways forward

Earth system models must predict forest responses to global change in order to simulate future global climate, hydrology, and ecosystem dynamics. These models are increasingly adopting vegetation demographic approaches that explicitly represent tree growth, mortality, and recruitment, enabling advances in the projection of forest vulnerability and resilience, as well as evaluation with field data. To date, simulation of regeneration processes has received far less attention than simulation of processes that affect growth and mortality, in spite of their critical role maintaining forest structure, facilitating turnover in forest composition over space and time, enabling recovery from disturbance, and regulating climate-driven range shifts. Here, our critical review of regeneration process representations within current Earth system vegetation demographic models reveals the need to improve parameter values and algorithms for reproductive allocation, dispersal, seed survival and germination, environmental filtering in the seedling layer, and tree regeneration strategies adapted to wind, fire, and anthropogenic disturbance regimes. These improvements require synthesis of existing data, specific field data-collection protocols, and novel model algorithms compatible with global-scale simulations. Vegetation demographic models offer the opportunity to more fully integrate ecological understanding into Earth system prediction; regeneration processes need to be a critical part of the effort.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2013

Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the coexpression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications.

09 BIOMASS FUELS↗
DOE OSTI2015

Metabolic engineering of oilseed crops to produce high levels of novel acetyl glyceride oils with reduced viscosity, freezing point and calorific value

Summary Seed oils have proved recalcitrant to modification for the production of industrially useful lipids. Here, we demonstrate the successful metabolic engineering and subsequent field production of an oilseed crop with the highest accumulation of unusual oil achieved so far in transgenic plants. Previously, expression of the E uonymus alatus diacylglycerol acetyltransferase ( E a DA c T ) gene in wild‐type A rabidopsis seeds resulted in the accumulation of 45 mol% of unusual 3‐acetyl‐1,2‐diacyl‐ sn ‐glycerols (acetyl‐ TAG s) in the seed oil (Durrett et al ., 2010 PNAS 107:9464). Expression of E a DA c T in dgat1 mutants compromised in their ability to synthesize regular triacylglycerols increased acetyl‐ TAG s to 65 mol%. Camelina and soybean transformed with the E a DA c T gene accumulate acetyl‐triacylglycerols (acetyl‐ TAG s) at up to 70 mol% of seed oil. A similar strategy of coexpression of E a DA c T together with RNA i suppression of DGAT 1 increased acetyl‐ TAG levels to up to 85 mol% in field‐grown transgenic C amelina. Additionally, total moles of triacylglycerol ( TAG ) per seed increased 20%. Analysis of the acetyl‐ TAG fraction revealed a twofold reduction in very long chain fatty acids ( VLCFA ), consistent with their displacement from the sn ‐3 position by acetate. Seed germination remained high, and seedlings were able to metabolize the stored acetyl‐ TAG s as rapidly as regular triacylglycerols. Viscosity, freezing point and caloric content of the C amelina acetyl‐ TAG oils were reduced, enabling use of this oil in several nonfood and food applications.

Liu, Jinjie↗
DOE OSTI2017

Identification of bottlenecks in the accumulation of cyclic fatty acids in camelina seed oil

Modified fatty acids (mFA) have diverse uses, e.g., cyclopropane fatty acids (CPA) are feedstocks for producing coatings, lubricants, plastics, and cosmetics. The expression of mFA-producing enzymes in crop and model plants generally results in lower levels of mFA accumulation than in their natural-occurring source plants. In order to further our understanding of metabolic bottlenecks that limit mFA accumulation, we generated transgenic Camelina sativa lines co-expressing Escherichia coli cyclopropane synthase (EcCPS) and Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT). In contrast to transgenic CPA-accumulating Arabidopsis, CPA accumulation in camelina caused only minor changes in seed weight, germination rate, oil accumulation, and seedling development. CPA accumulated to much higher levels in membrane than storage lipids, comprising more than 60% of total fatty acid in both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) versus 26% in diacylglycerol (DAG) and 12% in triacylglycerol (TAG) indicating bottlenecks in the transfer of CPA from PC to DAG and from DAG to TAG. Upon coexpression of SfLPAT with EcCPS, di-CPA-PC increased by ~50% relative to lines expressing EcCPS alone with the di-CPA-PC primarily observed in the embryonic axis and mono-CPA-PC primarily in cotyledon tissue. EcCPS-SfLPAT lines revealed a redistribution of CPA from the sn-1 to sn-2 positions within PC and PE that was associated with a doubling of CPA accumulation in both DAG and TAG. Finally, the identification of metabolic bottlenecks in acyl transfer between site of synthesis (phospholipids) and deposition in storage oils (TAGs) lays the foundation for the optimizing CPA accumulation through directed engineering of oil synthesis in target crops.

59 BASIC BIOLOGICAL SCIENCES↗