First draft reference genome and annotation of the alternative oil species Physaria fendleri.

In the wake of increasing demand for renewable energy sources, plant-based sources including alternative oilseeds have come to the forefront of interest. Hydroxy fatty acids (HFAs), produced in a few oilseed species, are important chemical feedstocks for industrial applications. An integrated approach was taken to assemble the first draft genome of the alternative HFA producer Physaria fendleri (n = 6), an outcrossing species with high heterozygosity. Both de novo transcriptome assemblies and genome assemblies were produced with public and generated sequencing reads. Resulting intermediate assemblies were then scaffolded and patched with multiple data sources, followed by super-scaffolding onto a masked genome of Camelina laxa (n = 6). Despite a current lack of available resources for the physical mapping of genomic scaffolds of Physaria fendleri, topography of the genome with respect to repeat and gene content was preserved at the scaffold level and not significantly lost via super-scaffolding. Read representation, gene and genome completion statistics, and annotation results illustrated the creation of a functional draft genome and a tool for future research on alternative oil species.

13C-labeling reveals non-conventional pathways providing carbon for hydroxy fatty acid synthesis in Physaria fendleri.

Physaria fendleri is a member of the Brassicaceae that produces in its embryos hydroxy fatty acids, constituents of oils that are very valuable and widely used by industry for cosmetics, lubricants, biofuels, etc. Free of toxins and rich in hydroxy fatty acids, Physaria provides a promising alternative to imported castor oil and is on the verge of being commercialized. This study aims to identify important biochemical step(s) for oil synthesis in Physaria, which may serve as target(s) for future crop improvement. To advance towards this goal, the endosperm composition was analysed by LC-MS/MS to develop and validate culture conditions that mimic the development of the embryos in planta. Using developing Physaria embryos in culture and 13C-labeling, our studies revealed that: (i) Physaria embryos metabolize carbon into biomass with an efficiency significantly lower than other photosynthetic embryos; (ii) the plastidic malic enzyme provides 42% of the pyruvate used for de novo fatty acid synthesis, which is the highest measured so far in developing 'green' oilseed embryos; and (iii) Physaria uses non-conventional pathways to channel carbon into oil, namely the Rubisco shunt, which fixes CO2 released in the plastid, and the reversibility of isocitrate dehydrogenase, which provides additional carbon for fatty acid elongation.

Time-resolved systems analysis of the induction of high photosynthetic capacity in Arabidopsis during acclimation to high light.

Induction of high photosynthetic capacity is a key acclimation response to high light (HL) for many herbaceous dicot plants; however, the signaling pathways that control this response remain largely unknown. Here, a systems biology approach was utilized to characterize the induction of high photosynthetic capacity in strongly and weakly acclimating Arabidopsis thaliana accessions. Plants were grown for 5 wk in a low light (LL) regime, and time-resolved photosynthetic physiological, metabolomic, and transcriptomic responses were measured during subsequent exposure to HL. The induction of high nitrogen (N) assimilation rates early in the HL shift was strongly predictive of the induction of photosynthetic capacity later in the HL shift. Accelerated N assimilation rates depended on the mobilization of existing organic acid (OA) reserves and increased de novo OA synthesis during the induction of high photosynthetic capacity. Enhanced sucrose biosynthesis capacity increased in tandem with the induction of high photosynthetic capacity, and increased starch biosynthetic capacity was balanced by increased starch catabolism. This systems analysis supports a model in which the efficient induction of N assimilation early in the HL shift begins the cascade of events necessary for the induction of high photosynthetic capacity acclimation in HL.

Metabolic and transcriptomic study of pennycress natural variation identifies targets for oil improvement.

Pennycress (Thlaspi arvense L.), a member of the Brassicaceae family, produces seed oil high in erucic acid, suitable for biodiesel and aviation fuel. Although pennycress, a winter annual, could be grown as a dedicated bioenergy crop, an increase in its seed oil content is required to improve its economic competitiveness. The success of crop improvement relies upon finding the right combination of biomarkers and targets, and the best genetic engineering and/or breeding strategies. In this work, we combined biomass composition with metabolomic and transcriptomic studies of developing embryos from 22 pennycress natural variants to identify targets for oil improvement. The selected accession collection presented diverse levels of fatty acids at maturity ranging from 29% to 41%. Pearson correlation analyses, weighted gene co-expression network analysis and biomarker identifications were used as complementary approaches to detect associations between metabolite level or gene expression and oil content at maturity. The results indicated that improving seed oil content can lead to a concomitant increase in the proportion of erucic acid without affecting the weight of embryos. Processes, such as carbon partitioning towards the chloroplast, lipid metabolism, photosynthesis, and a tight control of nitrogen availability, were found to be key for oil improvement in pennycress. Besides identifying specific targets, our results also provide guidance regarding the best timing for their modification, early or middle maturation. Thus, this work lays out promising strategies, specific for pennycress, to accelerate the successful development of lines with increased seed oil content for biofuel applications.

Progress in understanding and improving oil content and quality in seeds.

The world's population is projected to increase by two billion by 2050, resulting in food and energy insecurity. Oilseed crops have been identified as key to address these challenges: they produce and store lipids in the seeds as triacylglycerols that can serve as a source of food/feed, renewable fuels, and other industrially-relevant chemicals. Therefore, improving seed oil content and composition has generated immense interest. Research efforts aiming to unravel the regulatory pathways involved in fatty acid synthesis and to identify targets for metabolic engineering have made tremendous progress. This review provides a summary of the current knowledge of oil metabolism and discusses how photochemical activity and unconventional pathways can contribute to high carbon conversion efficiency in seeds. It also highlights the importance of 13C-metabolic flux analysis as a tool to gain insights on the pathways that regulate oil biosynthesis in seeds. Finally, a list of key genes and regulators that have been recently targeted to enhance seed oil production are reviewed and additional possible targets in the metabolic pathways are proposed to achieve desirable oil content and quality.

A lipidomics platform to analyze the fatty acid compositions of non-polar and polar lipid molecular species from plant tissues: Examples from developing seeds and seedlings of pennycress (Thlaspi arvense).

The lipidome comprises the total content of molecular species of each lipid class, and is measured using the analytical techniques of lipidomics. Many liquid chromatography-mass spectrometry (LC-MS) methods have previously been described to characterize the lipidome. However, many lipidomic approaches may not fully uncover the subtleties of lipid molecular species, such as the full fatty acid (FA) composition of certain lipid classes. Here, we describe a stepwise targeted lipidomics approach to characterize the polar and non-polar lipid classes using complementary LC-MS methods. Our "polar" method measures 260 molecular species across 12 polar lipid classes, and is performed using hydrophilic interaction chromatography (HILIC) on a NH2 column to separate lipid classes by their headgroup. Our "non-polar" method measures 254 molecular species across three non-polar lipid classes, separating molecular species on their FA characteristics by reverse phase (RP) chromatography on a C30 column. Five different extraction methods were compared, with an MTBE-based extraction chosen for the final lipidomics workflow. A state-of-the-art strategy to determine and relatively quantify the FA composition of triacylglycerols is also described. This lipidomics workflow was applied to developing, mature, and germinated pennycress seeds/seedlings and found unexpected changes among several lipid molecular species. During development, diacylglycerols predominantly contained long chain length FAs, which contrasted with the very long chain FAs of triacylglycerols in mature seeds. Potential metabolic explanations are discussed. The lack of very long chain fatty acids in diacylglycerols of germinating seeds may indicate very long chain FAs, such as erucic acid, are preferentially channeled into beta-oxidation for energy production.

Effective Mechanisms for Improving Seed Oil Production in Pennycress (Thlaspi arvense L.) Highlighted by Integration of Comparative Metabolomics and Transcriptomics.

Pennycress is a potentially lucrative biofuel crop due to its high content of long-chain unsaturated fatty acids, and because it uses non-conventional pathways to achieve efficient oil production. However, metabolic engineering is required to improve pennycress oilseed content and make it an economically viable source of aviation fuel. Research is warranted to determine if further upregulation of these non-conventional pathways could improve oil production within the species even more, which would indicate these processes serve as promising metabolic engineering targets and could provide the improvement necessary for economic feasibility of this crop. To test this hypothesis, we performed a comparative biomass, metabolomic, and transcriptomic analyses between a high oil accession (HO) and low oil accession (LO) of pennycress to assess potential factors required to optimize oil content. An evident reduction in glycolysis intermediates, improved oxidative pentose phosphate pathway activity, malate accumulation in the tricarboxylic acid cycle, and an anaplerotic pathway upregulation were noted in the HO genotype. Additionally, higher levels of threonine aldolase transcripts imply a pyruvate bypass mechanism for acetyl-CoA production. Nucleotide sugar and ascorbate accumulation also were evident in HO, suggesting differential fate of associated carbon between the two genotypes. An altered transcriptome related to lipid droplet (LD) biosynthesis and stability suggests a contribution to a more tightly-packed LD arrangement in HO cotyledons. In addition to the importance of central carbon metabolism augmentation, alternative routes of carbon entry into fatty acid synthesis and modification, as well as transcriptionally modified changes in LD regulation, are key aspects of metabolism and storage associated with economically favorable phenotypes of the species.

Natural variation and improved genome annotation of the emerging biofuel crop field pennycress (Thlaspi arvense).

The Brassicaceae family comprises more than 3,700 species with a diversity of phenotypic characteristics, including seed oil content and composition. Recently, the global interest in Thlaspi arvense L. (pennycress) has grown as the seed oil composition makes it a suitable source for biodiesel and aviation fuel production. However, many wild traits of this species need to be domesticated to make pennycress ideal for cultivation. Molecular breeding and engineering efforts require the availability of an accurate genome sequence of the species. Here, we describe pennycress genome annotation improvements, using a combination of long- and short-read transcriptome data obtained from RNA derived from embryos of 22 accessions, in addition to public genome and gene expression information. Our analysis identified 27,213 protein-coding genes, as well as on average 6,188 biallelic SNPs. In addition, we used the identified SNPs to evaluate the population structure of our accessions. The data from this analysis support that the accession Ames 32872, originally from Armenia, is highly divergent from the other accessions, while the accessions originating from Canada and the United States cluster together. When we evaluated the likely signatures of natural selection from alternative SNPs, we found 7 candidate genes under likely recent positive selection. These genes are enriched with functions related to amino acid metabolism and lipid biosynthesis and highlight possible future targets for crop improvement efforts in pennycress.

Dynamic nutrient acquisition from a hydrated apoplast supports biotrophic proliferation of a bacterial pathogen of maize.

Plant pathogens perturb their hosts to create environments suitable for their proliferation, including the suppression of immunity and promotion of water and nutrient availability. Although necrotrophs obtain water and nutrients by disrupting host-cell integrity, it is unknown whether hemibiotrophs, such as the bacterial pathogen Pantoea stewartii subsp. stewartii (Pnss), actively liberate water and nutrients during the early, biotrophic phase of infection. Here, we show that water and metabolite accumulation in the apoplast of Pnss-infected maize leaves precedes the disruption of host-cell integrity. Nutrient acquisition during this biotrophic phase is a dynamic process; the partitioning of metabolites into the apoplast rate limiting for their assimilation by proliferating Pnss cells. The formation of a hydrated and nutritive apoplast is driven by an AvrE-family type III effector, WtsE. Given the broad distribution of AvrE-family effectors, this work highlights the importance of actively acquiring water and nutrients for the proliferation of phytopathogenic bacteria during biotrophy.

Expression of AtWRI1 and AtDGAT1 during soybean embryo development influences oil and carbohydrate metabolism.

Soybean oil is one of the most consumed vegetable oils worldwide. Genetic improvement of its concentration in seeds has been historically pursued due to its direct association with its market value. Engineering attempts aiming to increase soybean seed oil presented different degrees of success that varied with the genetic design and the specific variety considered. Understanding the embryo's responses to the genetic modifications introduced, is a critical step to successful approaches. In this work, the metabolic and transcriptional responses to AtWRI1 and AtDGAT1 expression in soybean seeds were evaluated. AtWRI1 is a master regulator of fatty acid (FA) biosynthesis, and AtDGAT1 encodes an enzyme catalysing the final and rate-limiting step of triacylglycerides biosynthesis. The events expressing these genes in the embryo did not show an increase in total FA content, but they responded with changes in the oil and carbohydrate composition. Transcriptomic studies revealed a down-regulation of genes putatively encoding for oil body packaging proteins, and a strong induction of genes annotated as lipases and FA biosynthesis inhibitors. Novel putative AtWRI1 targets, presenting an AW-box in the upstream region of the genes, were identified by comparison with an event that harbours only AtWRI1. Lastly, targeted metabolomics analysis showed that carbon from sugar phosphates could be used for FA competing pathways, such as starch and cell wall polysaccharides, contributing to the restriction in oil accumulation. These results allowed the identification of key cellular processes that need to be considered to break the embryo's natural restriction to uncontrolled seed lipid increase.

Metabolite shift in Medicago truncatula occurs in phosphorus deprivation.

Symbiotic nitrogen (N) fixation entails successful interaction between legume hosts and rhizobia that occur in specialized organs called nodules. N-fixing legumes have a higher demand for phosphorus (P) than legumes grown on mineral N. Medicago truncatula is an important model plant for characterization of effects of P deficiency at the molecular level. Hence, a study was carried out to address the alteration in metabolite levels of M. truncatula grown aeroponically and subjected to 4 weeks of P stress. First, GC-MS-based untargeted metabolomics initially revealed changes in the metabolic profile of nodules, with increased levels of amino acids and sugars and a decline in amounts of organic acids. Subsequently, LC-MS/MS was used to quantify these compounds including phosphorylated metabolites in the whole plant. Our results showed a drastic reduction in levels of organic acids and phosphorylated compounds in -P leaves, with a moderate reduction in -P roots and nodules. Additionally, sugars and amino acids were elevated in the whole plant under P deprivation. These findings provide evidence that N fixation in M. truncatula is mediated through a N feedback mechanism that in parallel is related to carbon and P metabolism.

A metabolomic platform to identify and quantify polyphenols in coffee and related species using liquid chromatography mass spectrometry.

Introduction: Products of plant secondary metabolism, such as phenolic compounds, flavonoids, alkaloids, and hormones, play an important role in plant growth, development, stress resistance. The plant family Rubiaceae is extremely diverse and abundant in Central America and contains several economically important genera, e.g. Coffea and other medicinal plants. These are known for the production of bioactive polyphenols (e.g. caffeine and quinine), which have had major impacts on human society. The overall goal of this study was to develop a high-throughput workflow to identify and quantify plant polyphenols. Methods: First, a method was optimized to extract over 40 families of phytochemicals. Then, a high-throughput metabolomic platform has been developed to identify and quantify 184 polyphenols in 15 min. Results: The current metabolomics study of secondary metabolites was conducted on leaves from one commercial coffee variety and two wild species that also belong to the Rubiaceae family. Global profiling was performed using liquid chromatography high-resolution time-of-flight mass spectrometry. Features whose abundance was significantly different between coffee species were discriminated using statistical analysis and annotated using spectral databases. The identified features were validated by commercially available standards using our newly developed liquid chromatography tandem mass spectrometry method. Discussion: Caffeine, trigonelline and theobromine were highly abundant in coffee leaves, as expected. Interestingly, wild Rubiaceae leaves had a higher diversity of phytochemicals in comparison to commercial coffee: defense-related molecules, such as phenylpropanoids (e.g., cinnamic acid), the terpenoid gibberellic acid, and the monolignol sinapaldehyde were found more abundantly in wild Rubiaceae leaves.

The Evolution of Leaf Function during Development Is Reflected in Profound Changes in the Metabolic Composition of the Vacuole.

During its development, the leaf undergoes profound metabolic changes to ensure, among other things, its growth. The subcellular metabolome of tomato leaves was studied at four stages of leaf development, with a particular emphasis on the composition of the vacuole, a major actor of cell growth. For this, leaves were collected at different positions of the plant, corresponding to different developmental stages. Coupling cytology approaches to non-aqueous cell fractionation allowed to estimate the subcellular concentrations of major compounds in the leaves. The results showed major changes in the composition of the vacuole across leaf development. Thus, sucrose underwent a strong allocation, being mostly located in the vacuole at the beginning of development and in the cytosol at maturity. Furthermore, these analyses revealed that the vacuole, rather rich in secondary metabolites and sugars in the growth phases, accumulated organic acids thereafter. This result suggests that the maintenance of the osmolarity of the vacuole of mature leaves would largely involve inorganic molecules.

Analyzing Mass Spectrometry Imaging Data of 13C-Labeled Phospholipids in Camelina sativa and Thlaspi arvense (Pennycress) Embryos.

The combination of 13C-isotopic labeling and mass spectrometry imaging (MSI) offers an approach to analyze metabolic flux in situ. However, combining isotopic labeling and MSI presents technical challenges ranging from sample preparation, label incorporation, data collection, and analysis. Isotopic labeling and MSI individually create large, complex data sets, and this is compounded when both methods are combined. Therefore, analyzing isotopically labeled MSI data requires streamlined procedures to support biologically meaningful interpretations. Using currently available software and techniques, here we describe a workflow to analyze 13C-labeled isotopologues of the membrane lipid and storage oil lipid intermediate-phosphatidylcholine (PC). Our results with embryos of the oilseed crops, Camelina sativa and Thlaspi arvense (pennycress), demonstrated greater 13C-isotopic labeling in the cotyledons of developing embryos compared with the embryonic axis. Greater isotopic enrichment in PC molecular species with more saturated and longer chain fatty acids suggest different flux patterns related to fatty acid desaturation and elongation pathways. The ability to evaluate MSI data of isotopically labeled plant embryos will facilitate the potential to investigate spatial aspects of metabolic flux in situ.

Targeted Metabolic Profiles of the Leaves and Xylem Sap of Two Sugarcane Genotypes Infected with the Vascular Bacterial Pathogen Leifsonia xyli subsp. xyli.

Ratoon stunt (RS) is a worldwide disease that reduces biomass up to 80% and is caused by the xylem-dwelling bacterium Leifsonia xyli subsp. xyli. This study identified discriminant metabolites between a resistant (R) and a susceptible (S) sugarcane variety at the early stages of pathogen colonization (30 and 120 days after inoculation-DAI) by untargeted and targeted metabolomics of leaves and xylem sap using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. Bacterial titers were quantified in sugarcane extracts at 180 DAI through real-time polymerase chain reaction. Bacterial titers were at least four times higher on the S variety than in the R one. Global profiling detected 514 features in the leaves and 68 in the sap, while 119 metabolites were quantified in the leaves and 28 in the sap by targeted metabolomics. Comparisons between mock-inoculated treatments indicated a greater abundance of amino acids in the leaves of the S variety and of phenolics, flavonoids, and salicylic acid in the R one. In the xylem sap, fewer differences were detected among phenolics and flavonoids, but also included higher abundances of the signaling molecule sorbitol and glycerol in R. Metabolic changes in the leaves following pathogen inoculation were detected earlier in R than in S and were mostly related to amino acids in R and to phosphorylated compounds in S. Differentially represented metabolites in the xylem sap included abscisic acid. The data represent a valuable resource of potential biomarkers for metabolite-assisted selection of resistant varieties to RS.

Phosphorus deprivation affects composition and spatial distribution of membrane lipids in legume nodules.

In legumes, symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. In a nodule, N-fixing rhizobia are surrounded by symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N2-fixing legumes have a higher requirement for P than legumes grown on mineral N. As in the previous studies, in P deficiency, barrel medic (Medicago truncatula) plants had impaired SNF activity, reduced growth, and accumulated less phosphate in leaves, roots, and nodules compared with the plants grown in P sufficient conditions. Membrane lipids in M. truncatula tissues were assessed using electrospray ionization-mass spectrometry. Galactolipids were found to increase in P deficiency, with declines in phospholipids (PL), especially in leaves. Lower PL losses were found in roots and nodules. Subsequently, matrix-assisted laser desorption/ionization-mass spectrometry imaging was used to spatially map the distribution of the positively charged phosphatidylcholine (PC) species in nodules in both P-replete and P-deficient conditions. Our results reveal heterogeneous distribution of several PC species in nodules, with homogeneous distribution of other PC classes. In P poor conditions, some PC species distributions were observed to change. The results suggest that specific PC species may be differentially important in diverse nodule zones and cell types, and that membrane lipid remodeling during P stress is not uniform across the nodule.

Non-conventional pathways enable pennycress (Thlaspi arvense L.) embryos to achieve high efficiency of oil biosynthesis.

Pennycress (Thlaspi arvense L.) accumulates oil up to 35% of the total seed biomass, and its overall fatty acid composition is suitable for aviation fuel. However, for this plant to become economically viable, its oil production needs to be improved. In vivo culture conditions that resemble the development of pennycress embryos in planta were developed based on the composition of the liquid endosperm. Then, substrate uptake rates and biomass accumulation were measured from cultured pennycress embryos, revealing a biosynthetic efficiency of 93%, which is one of the highest in comparison with other oilseeds to date. Additionally, the ratio of carbon in oil to CO2 indicated that non-conventional pathways are likely to be responsible for such a high carbon conversion efficiency. To identify the reactions enabling this phenomenon, parallel labeling experiments with 13C-labeled substrates were conducted in pennycress embryos. The main findings of these labeling experiments include: (i) the occurrence of the oxidative reactions of the pentose phosphate pathway in the cytosol; (ii) the reversibility of isocitrate dehydrogenase; (iii) the operation of the plastidic NADP-dependent malic enzyme; and (iv) the refixation of CO2 by Rubisco. These reactions are key providers of carbon and reductant for fatty acid synthesis and elongation.

A Combined Metabolomics and Fluxomics Analysis Identifies Steps Limiting Oil Synthesis in Maize Embryos.

Enhancing fatty acid synthesis (FAS) in maize (Zea mays) has tremendous potential nutritional and economic benefits due to the rapidly growing demand for vegetable oil. In maize kernels, the endosperm and the embryo are the main sites for synthesis and accumulation of starch and oil, respectively. So far, breeding efforts to achieve elevated oil content in maize have resulted in smaller endosperms and therefore lower yield. Directly changing their carbon metabolism may be the key to increasing oil content in maize kernels without affecting yield. To test this hypothesis, the intracellular metabolite levels were compared in maize embryos from two different maize lines, ALEXHO S K SYNTHETIC (Alex) and LH59, which accumulate 48% and 34% of oil, respectively. Comparative metabolomics highlighted the metabolites and pathways that were active in the embryos and important for oil production. The contribution of each pathway to FAS in terms of carbon, reductant, and energy provision was assessed by measuring the carbon flow through the metabolic network (13C-metabolic flux analysis) in developing Alex embryos to build a map of carbon flow through the central metabolism. This approach combined mathematical modeling with biochemical quantification to identify metabolic bottlenecks in FAS in maize embryos. This study describes a combination of innovative tools that will pave the way for controlling seed composition in important food crops.

A Simple Method for Measuring Apoplast Hydration and Collecting Apoplast Contents.

The plant leaf apoplast is a dynamic environment subject to a variety of both internal and external stimuli. In addition to being a conduit for water vapor and gas exchange involved in transpiration and photosynthesis, the apoplast also accumulates many nutrients transported from the soil as well as those produced through photosynthesis. The internal leaf also provides a protective environment for endophytic and pathogenic microbes alike. Given the diverse array of physiological processes occurring in the apoplast, it is expedient to develop methods to study its contents. Many established methods rely on vacuum infiltration of an apoplast wash solution followed by centrifugation. In this study, we describe a refined method optimized for maize (Zea mays) seedling leaves, which not only provides a simple procedure for obtaining apoplast fluid, but also allows direct calculation of apoplast hydration at the time of harvest for every sample. In addition, we describe an abbreviated method for estimating apoplast hydration if the full apoplast extraction is not necessary. Finally, we show the applicability of this optimized apoplast extraction procedure for plants infected with the maize pathogen Pantoea stewartii ssp stewartii, including the efficient isolation of bacteria previously residing in the apoplast. The approaches to establishing this method should make it generally applicable to other types of plants.