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DOE OSTI2023

Exopolysaccharide is required for motility, stress tolerance, and plant colonization by the endophytic bacterium Paraburkholderia phytofirmans PsJN

Paraburkholderia phytofirmans PsJN is an endophytic bacterium and has been shown to promote the growth and health of many different plants. Exopolysaccharide (EPS) plays important roles in plant-bacteria interaction and tolerance to environmental stresses. However, the function of EPS in PsJN and its interaction with plants remain largely unknown. In this study, a deletion mutation of bceQ gene, encoding a putative flippase for the EPS biosynthesis, was introduced in the genome of PsJN. The ΔbceQ mutant produced a significantly lower level of EPS than the wild type strain in culture media. Compared to the wild type PsJN, the ΔbceQ mutant was more sensitive to desiccation, UV damage, salt (NaCl) and iron (FeCl 3 ) stresses, and bacteriophage infection. More importantly, the mutation of bceQ decreased the endophytic colonization of PsJN in camelina (Camelina sativa) and pea (Camelina sativa) under plant drought stress conditions. To the best of our knowledge, this is the first report that EPS production is required for the maximal colonization of an endophytic bacterium in the plant tissues under stress conditions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Generation of camelina mid-oleic acid seed oil by identification and stacking of fatty acid biosynthetic mutants

Modifying oilseeds to obtain a desired fatty acid composition is often necessary to enable use as feedstocks for specific applications such as food processing, biofuels, or biolubricants. A mutant population of camelina (Camelina sativa), an emerging specialty oilseed crop, was screened by high-throughput gas chromatography for lines with altered seed oil fatty acid composition. By leveraging knowledge of fatty acid synthesis in Arabidopsis thaliana, mutations in orthologs of FATTY ACID ELONGASE1 (FAE1), FATTY ACID DESATURASE2 (FAD2), FATTY ACID DESATURASE3 (FAD3), and β-KETO-ACYL-ACP SYNTHASE II (KASII; FAB1) were identified. The mutations altered conserved amino acid residues in the encoded proteins. The ability of the mutations in FAE1, FAD2 and FAD3 to affect enzyme function was demonstrated by comparing in vivo activities of wild-type and mutant alleles in yeast. In addition, expression of wild-type cDNA in camelina complemented fatty acid phenotypes of these mutants. As camelina has a hexaploid genome, the effect of a mutation in one of the three homeologs for each gene resulted in no or less severe growth phenotypes compared to similar mutations in Arabidopsis. Mid-oleic oils with nearly 40 % oleic acid and reduced very long-chain (≤C20) fatty acid content were obtained by crossing to obtain a fae1c/fad2a/fae1a/fad3a quadruple mutant. Little effect on total seed oil content was observed in the stacked mutant line. Finally, the resulting mid-oleic acid oil had improved oxidative stability due to reductions in polyunsaturated fatty acid content, increasing its utility for biofuels and other applications.

09 BIOMASS FUELS↗

DOE OSTI2023

Daylength variation affects growth, photosynthesis, leaf metabolism, partitioning, and metabolic fluxes

Abstract Daylength, a seasonal and latitudinal variable, exerts a substantial impact on plant growth. However, the relationship between daylength and growth is nonproportional, suggesting the existence of adaptive mechanisms. Thus, our study aimed to comprehensively investigate the adaptive strategies employed by plants in response to daylength variation. We grew false flax (Camelina sativa) plants, a model oilseed crop, under long-day (LD) and short-day (SD) conditions and used growth measurements, gas exchange measurements, and isotopic labeling techniques, including 13C, 14C, and 2H2O, to determine responses to different daylengths. Our findings revealed that daylength influences various growth parameters, photosynthetic physiology, carbon partitioning, metabolic fluxes, and metabolite levels. SD plants employed diverse mechanisms to compensate for reduced CO2 fixation in the shorter photoperiod. These mechanisms included enhanced photosynthetic rates and reduced respiration in the light (RL), leading to increased shoot investment. Additionally, SD plants exhibited reduced rates of the glucose 6-phosphate (G6P) shunt and greater partitioning of sugars into starch, thereby sustaining carbon availability during the longer night. Isotopic labeling results further demonstrated substantial alterations in the partitioning of amino acids and TCA cycle intermediates between rapidly and slowly turning over pools. Overall, the results point to multiple developmental, physiological, and metabolic ways in which plants adapt to different daylengths to maintain growth.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Genetic dissection of natural variation in oilseed traits of camelina by whole‐genome resequencing and QTL mapping

Abstract Camelina [ Camelina sativa (L.) Crantz] is an oilseed crop in the Brassicaceae family that is currently being developed as a source of bioenergy and healthy fatty acids. To facilitate modern breeding efforts through marker‐assisted selection and biotechnology, we evaluated genetic variation among a worldwide collection of 222 camelina accessions. We performed whole‐genome resequencing to obtain single nucleotide polymorphism (SNP) markers and to analyze genomic diversity. We also conducted phenotypic field evaluations in two consecutive seasons for variations in key agronomic traits related to oilseed production such as seed size, oil content (OC), fatty acid composition, and flowering time. We determined the population structure of the camelina accessions using 161,301 SNPs. Further, we identified quantitative trait loci (QTL) and candidate genes controlling the above field‐evaluated traits by genome‐wide association studies (GWAS) complemented with linkage mapping using a recombinant inbred line (RIL) population. Characterization of the natural variation at the genome and phenotypic levels provides valuable resources to camelina genetic studies and crop improvement. The QTL and candidate genes should assist in breeding of advanced camelina varieties that can be integrated into the cropping systems for the production of high yield of oils of desired fatty acid composition.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Chromosome-level assembly and analysis of Camelina neglecta : a novel diploid model for Camelina biotechnology research

Camelina neglecta is a new diploid Brassicaceae species, which has great research value because of its close relationship with the hexaploid oilseed crop Camelina sativa. Here, we report a chromosome-level assembly of C. neglecta with a total length of 210 Mb. By adopting PacBio sequencing and Hi-C technology, the C. neglecta genome was assembled into 6 chromosomes with scaffold N50 of 29.62 Mb. C. neglecta has undergone the whole-genome triplication (γ) shared among eudicots and two whole-genome duplications (α and β) shared by crucifers, but it has not undergone a specific whole-genome duplication event. By synteny analysis between C. neglecta and C. sativa, we successfully used the method of calculating Ks to distinguish the three subgenomes of C. sativa and determined that C. neglecta was closest to the first subgenome (SG1) of C. sativa. Further, transcriptomic analysis revealed the key genes associated with seed oil biosynthesis and its transcriptional regulation, including SAD, FAD2, FAD3, FAE1, ABI3, WRI1 and FUS3 displaying high expression levels in C. neglecta seeds. The high representability of C. neglecta as a model species for Camelina-based biotechnology research has been demonstrated for the first time. In particular, floral Agrobacterium tumefaciens infiltration-based transformation of C. neglecta, leading to overexpression of CvLPAT2, CpDGAT1 and CvFatB1 transgenes, was demonstrated for medium-chain fatty acid accumulation in C. neglecta seed oil. This study provides an important genomic resource and establishes C. neglecta as a new model for oilseed biotechnology research.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Life Cycle Greenhouse Gas Emissions of Growing Intermediate Winter Oilseed Feedstocks for Sustainable Aviation Fuel Production

Ensuring an adequate supply of feedstock is a key challenge to achieving a significant production volume of sustainable aviation fuels (SAFs). One promising feedstock for SAFs is intermediate winter oilseeds added to existing crop production as alternatives to the winter fallow period. In this work, three intermediate winter oilseed crops─camelina ( Camelina sativa L. Crantz .), carinata ( Brassica carinata ), and pennycress ( Thlaspi arvense L .)─are evaluated for their life cycle greenhouse gas (GHG) emissions when harvested and processed into hydroprocessed renewable jet (HRJ). We evaluate solvent extraction (SE) and mechanical pressing as alternative oil extraction methods and examine the impacts of various allocation methods used in the oil extraction process. The GHG emissions reduction potential of camelina-, carinata-, and pennycress-derived HRJs are 50.4%, 65.2%, and 65.7%, respectively, compared to petroleum jet fuel, with SE as an oil extraction method and under mass-based allocation. Combined with the estimated production potential of camelina, carinata, and pennycress as intermediate winter oilseed, 2.5 billion gallons of HRJ can be produced annually, and the total GHG savings could amount to 19.3 million tons when these HRJs replace petroleum jet fuels. In addition, we collect literature information to assess the impacts of these intermediate winter oilseeds on indirect land use change emissions and soil organic carbon sequestration potentials.

camelina↗

DOE OSTI2021

Increased expression of fatty acid and ABC transporters enhances seed oil production in camelina

Abstract Background Lipid transporters play an essential role in lipid delivery and distribution, but their influence on seed oil production in oilseed crops is not well studied. Results Here, we examined the effect of two lipid transporters, FAX1 ( fatty acid export1 ) and ABCA9 ( ATP-binding cassette transporter subfamily A9 ) on oil production and lipid metabolism in the oilseed plant Camelina sativa . Overexpression (OE) of FAX1 and ABCA9 increased seed weight and size, with FAX1 -OEs and ABCA9 -OEs increasing seed length and width, respectively, whereas FAX1 / ABCA9 -OEs increasing both. FAX1- OE and ABCA9- OE displayed additive effects on seed oil content and seed yield. Also, OE of FAX1 and ABCA9 affected membrane lipid composition in developing pods, especially on phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol. The expression of some genes involved in seed oil synthesis, such as DGAT2 , PDAT1 , and LEC1 , was increased in developing seeds of FAX1 - and/or ABCA9 -OEs. Conclusion These results indicate that increased expression of FAX1 and ABCA9 can potentially be applied to improving camelina oil production.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Genome-wide identification and diversity of FAD2, FAD3 and FAE1 genes in terms of biotechnological importance in Camelina species

False flax, or gold-of-pleasure (Camelina sativa) is an oilseed that has received renewed research interest as a promising vegetable oil feedstock for liquid biofuel production and other non-food uses. This species has also emerged as a model for oilseed biotechnology research that aims to enhance seed oil content and fatty acid quality. To date, a number of genetic engineering and gene editing studies on C. sativa have been reported. Among the most common targets for this research are genes, encoding fatty acid desaturases, elongases, and diacylglycerol acyltransferases. However, the majority of these genes in C. sativa are present in multiple copies due to the allohexaploid nature of the species. Therefore, genetic manipulations require a comprehensive understanding of the diversity of such gene targets.

09 BIOMASS FUELS↗

DOE OSTI2025

Rapid quantification of whole seed fatty acid amount, composition, and shape phenotypes from diverse oilseed species with large differences in seed size

Seed oils are widely used in the food, biofuel, and industrial feedstock industries, with their utility and value determined by total oil content and fatty acid composition. Current high throughput seed oil analysis methods either lack accuracy in total fatty acid profiling or require extensive labor for lipid extraction prior to derivatization to fatty acid methyl esters (FAME) and quantification by gas chromatography (GC). Alternatively, direct whole seed FAME production methods have been developed for the very small seeds in the model species Arabidopsis thaliana but these have generally not been adapted to larger seeds of most oilseed crops. High-throughput direct whole seed FAME production methods were optimized for seeds up to 5 mg each utilizing acid-catalyzed esterification. For the oilseed species Camelina sativa, Thlaspi avernse (pennycress), Cuphea viscosissima, and Brassica napus (var. Canola), the total seed fatty acid content and composition from direct seed esterification to FAME matched that of lipid extract derivatization demonstrating the accuracy of the methods. In combination with seed phenotyping using GridFree, this approach enabled the development of a rapid pipeline for simultaneous seed weight, count, size/shape phenotyping, and oil analysis. For the larger and tougher seeds produced by Limnanthes alba (Meadowfoam) and Cannabis sativa L. (hemp) the whole seed acid-based method proved insufficient, and prior laborious homogenization of seeds was required. Therefore, a rapid one-tube bead homogenization and base catalyzed-esterification method was developed. Base-derived fatty acid esterification cannot derivatize free fatty acids leading to slightly lower total seed fatty acid than acid-catalyzed methods, however the seed oil content and fatty acid composition that is valuable for screening large numbers of samples in research populations was accurately measured. New rapid whole seed fatty acid esterification and phenotyping protocols were developed to accurately assess oilseed lipid content. These methods are particularly valuable in oilseed research, breeding, and engineering applications where efficient analysis of large numbers of samples and accurate oil fatty acid profiling is essential. While having been developed for current and emerging oilseed crops, these methods also provide a foundation from which protocols might be established for new and emerging crop species.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Evaluating the contribution of plant metabolic pathways in the light to the ATP:NADPH demand using a meta-analysis of isotopically non-stationary metabolic flux analyses

Abstract Balancing the ATP: NADPH demand from plant metabolism with supply from photosynthesis is essential for preventing photodamage and operating efficiently, so understanding its drivers is important for integrating metabolism with the light reactions of photosynthesis and for bioengineering efforts that may radically change this demand. It is often assumed that the C3 cycle and photorespiration consume the largest amount of ATP and reductant in illuminated leaves and as a result mostly determine the ATP: NADPH demand. However, the quantitative extent to which other energy consuming metabolic processes contribute in large ways to overall ATP: NADPH demand remains unknown. Here, we used the metabolic flux networks of numerous recently published isotopically non-stationary metabolic flux analyses (INST-MFA) to evaluate flux through the C3 cycle, photorespiration, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and starch/sucrose synthesis and characterize broad trends in the demand of energy across different pathways and compartments as well as in the overall ATP:NADPH demand. These data sets include a variety of species including Arabidopsis thaliana , Nicotiana tabacum , and Camelina sativa as well as varying environmental factors including high/low light, day length, and photorespiratory levels. Examining these datasets in aggregate reveals that ultimately the bulk of the energy flux occurred in the C3 cycle and photorespiration, however, the energy demand from these pathways did not determine the ATP: NADPH demand alone. Instead, a notable contribution was revealed from starch and sucrose synthesis which might counterbalance photorespiratory demand and result in fewer adjustments in mechanisms which balance the ATP deficit.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

The effects of photosynthetic rate on respiration in light, starch/sucrose partitioning, and other metabolic fluxes within photosynthesis

In the future, plants may encounter increased light and elevated CO 2 levels. How consequent alterations in photosynthetic rates will impact fluxes in photosynthetic carbon metabolism remains uncertain. Respiration in light ( R L ) is pivotal in plant carbon balance and a key parameter in photosynthesis models. Understanding the dynamics of photosynthetic metabolism and R L under varying environmental conditions is essential for optimizing plant growth and agricultural productivity. However, measuring R L under high light and high CO 2 (HLHC) conditions poses challenges using traditional gas exchange methods. In this study, we employed isotopically nonstationary metabolic flux analysis (INST-MFA) to estimate RL and investigate photosynthetic carbon flux, unveiling nuanced adjustments in Camelina sativa under HLHC. Despite numerous flux alterations in HLHC, RL remained stable. HLHC affects several factors influencing RL, such as starch and sucrose partitioning, v o /v c ratio, triose phosphate partitioning, and hexose kinase activity. Analysis of A/C i curve operational points reveals that HLHC’s major changes primarily stem from CO 2 suppressing photorespiration. Integration of these fluxes into a simplified model predicts changes in CBC labeling under HLHC. This study extends our prior discovery that incomplete CBC labeling is due to unlabeled carbon reimported during R L , offering insights into manipulating labeling through adjustments in photosynthetic rates.

Elevated CO2↗

DOE OSTI2024

FatPlants: a comprehensive information system for lipid-related genes and metabolic pathways in plants

Abstract FatPlants, an open-access, web-based database, consolidates data, annotations, analysis results, and visualizations of lipid-related genes, proteins, and metabolic pathways in plants. Serving as a minable resource, FatPlants offers a user-friendly interface for facilitating studies into the regulation of plant lipid metabolism and supporting breeding efforts aimed at increasing crop oil content. This web resource, developed using data derived from our own research, curated from public resources, and gleaned from academic literature, comprises information on known fatty-acid-related proteins, genes, and pathways in multiple plants, with an emphasis on Glycine max, Arabidopsis thaliana, and Camelina sativa. Furthermore, the platform includes machine-learning based methods and navigation tools designed to aid in characterizing metabolic pathways and protein interactions. Comprehensive gene and protein information cards, a Basic Local Alignment Search Tool search function, similar structure search capacities from AphaFold, and ChatGPT-based query for protein information are additional features. Database URL: https://www.fatplants.net/

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids

Abstract Assessing central carbon metabolism in plants can be challenging due to the dynamic range in pool sizes, with low levels of important phosphorylated sugars relative to more abundant sugars and organic acids. Here, we report a sensitive liquid chromatography–mass spectrometry method for analysing central metabolites on a hybrid column, where both anion-exchange and hydrophilic interaction chromatography (HILIC) ligands are embedded in the stationary phase. The liquid chromatography method was developed for enhanced selectivity of 27 central metabolites in a single run with sensitivity at femtomole levels observed for most phosphorylated sugars. The method resolved phosphorylated hexose, pentose, and triose isomers that are otherwise challenging. Compared with a standard HILIC approach, these metabolites had improved peak areas using our approach due to ion enhancement or low ion suppression in the biological sample matrix. The approach was applied to investigate metabolism in high lipid-producing tobacco leaves that exhibited increased levels of acetyl-CoA, a precursor for oil biosynthesis. The application of the method to isotopologue detection and quantification was considered through evaluating 13C-labeled seeds from Camelina sativa. The method provides a means to analyse intermediates more comprehensively in central metabolism of plant tissues.

Plant Sciences↗

DOE OSTI2021

The metabolic origins of non-photorespiratory CO 2 release during photosynthesis: a metabolic flux analysis

Respiration in the light (R L ) releases CO 2 in photosynthesizing leaves and is a phenomenon that occurs independently from photorespiration. Since R L lowers net carbon fixation, understanding R L could help improve plant carbon-use efficiency and models of crop photosynthesis. Although R L was identified more than 75 years ago, its biochemical mechanisms remain unclear. To identify reactions contributing to R L , we mapped metabolic fluxes in photosynthesizing source leaves of the oilseed crop and model plant camelina (Camelina sativa). We performed a flux analysis using isotopic labeling patterns of central metabolites during 13 CO 2 labeling time course, gas exchange, and carbohydrate production rate experiments. To quantify the contributions of multiple potential CO 2 sources with statistical and biological confidence, we increased the number of metabolites measured and reduced biological and technical heterogeneity by using single mature source leaves and quickly quenching metabolism by directly injecting liquid N 2 ; we then compared the goodness-of-fit between these data and data from models with alternative metabolic network structures and constraints. Our analysis predicted that R L releases 5.2 μmol CO2 g –1 FW h –1 of CO 2 , which is relatively consistent with a value of 9.3 μmol CO 2 g –1 FW h –1 measured by CO 2 gas exchange. The results indicated that ≤10% of R L results from TCA cycle reactions, which are widely considered to dominate R L . Further analysis of the results indicated that oxidation of glucose-6-phosphate to pentose phosphate via 6-phosphogluconate (the G6P/OPP shunt) can account for >% of CO 2 released by R L .

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

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

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

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

DOE OSTI2022

High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes

Abstract Background Genetic engineering of crop plants has been successful in transferring traits into elite lines beyond what can be achieved with breeding techniques. Introduction of transgenes originating from other species has conferred resistance to biotic and abiotic stresses, increased efficiency, and modified developmental programs. The next challenge is now to combine multiple transgenes into elite varieties via gene stacking to combine traits. Generating stable homozygous lines with multiple transgenes requires selection of segregating generations which is time consuming and labor intensive, especially if the crop is polyploid. Insertion site effects and transgene copy number are important metrics for commercialization and trait efficiency. Results We have developed a simple method to identify the sites of transgene insertions using T-DNA-specific primers and high-throughput sequencing that enables identification of multiple insertion sites in the T 1 generation of any crop transformed via Agrobacterium . We present an example using the allohexaploid oil-seed plant Camelina sativa to determine insertion site location of two transgenes. Conclusion This new methodology enables the early selection of desirable transgene location and copy number to generate homozygous lines within two generations.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

A Systems Framework to Enhance the Potential of Camelina as Oilseed Crop (Final Report)

To alleviate the dependency of the United States on fossil fuels, particularly from foreign sources, the transition to biofuel crops has long been proposed as a crucial part of the long-term solution. Camelina sativa has emerged as one of the most promising oilseed crops for domestic biofuel and bioproduct production due to its low agronomic input requirements, natural resilience to a wide range of biotic and abiotic stresses, and the successful testing and approval of its oil-based blends for use in liquid transportation fuels.

09 BIOMASS FUELS↗

NASA NTRS2015

Effects of Burning Alternative Fuel in a 5-Cup Combustor Sector

A goal of NASA's Environmentally Responsible Aviation (ERA) program is to develop a combustor that will reduce the NOx emissions and that can burn both standard and alternative fuels. To meet this goal, NASA partnered with General Electric Aviation to develop a 5-cup combustor sector; this sector was tested in NASA Glenn's Advanced Subsonic Combustion Rig (ASCR). To verify that the combustor sector was fuel-flexible, it was tested with a 50-50 blend of JP-8 and a biofuel made from the camelina sativa plant. Results from this test were compared to results from tests where the fuel was neat JP-8. Testing was done at three combustor inlet conditions: cruise, 30% power, and 7% power. When compared to burning JP-8, burning the 50-50 blend did not significantly affect emissions of NOx, CO, or total hydrocarbons. Furthermore, it did not significantly affect the magnitude and frequency of the dynamic pressure fluctuations.

alternative fuel↗