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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 13 C-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.

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.

Summary 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.

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 feed stocks 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 P. 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.

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.

Summary 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.

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 is extremely diverse and abundant in Central America and contains several economically important genera, e.g. 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. 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. 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 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. Caffeine, trigonelline and theobromine were highly abundant in coffee leaves, as expected. Interestingly, wild 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.

ABSTRACT Objectives: to know the therapeutic itinerary and to show the implications of breast cancer for women in a border town in southern Brazil. Methods: a qualitative research conducted based on the complexity paradigm, through interviews with 13 women with breast cancer undergoing outpatient treatment and data analyzed by thematic analysis. Results: the first signs and symptoms of breast cancer were noticed unexpectedly, in everyday moments and during routine visits. There was family support in the search for assistance in primary care services, but organizational barriers led this itinerary to private health services, including Paraguay. Coping with the disease and treatment was anchored in the family subsystem. Final Considerations: the paths for breast cancer diagnosis and treatment included families, primary care services and private health services, including from the neighboring country, considering the vulnerability of border regions to guarantee health care. RESUMEN Objetivos: conocer el itinerario terapéutico y mostrar las implicaciones del cáncer de mama para las mujeres de una localidad fronteriza del sur de Brasil. Métodos: investigación cualitativa, realizada con base en el paradigma de la complejidad, a través de entrevistas a 13 mujeres con cáncer de mama en tratamiento ambulatorio y datos analizados mediante análisis temático. Resultados: los primeros signos y síntomas del cáncer de mama se notaron de forma inesperada, en los momentos cotidianos y durante las visitas de rutina. Hubo apoyo familiar en la búsqueda de asistencia en los servicios de Atención Primaria, pero barreras organizacionales llevaron este itinerario a los servicios privados de salud, incluido Paraguay. El afrontamiento de la enfermedad y el tratamiento estaba anclado en el subsistema familiar. Consideraciones Finales: los caminos para el diagnóstico y tratamiento del cáncer de mama incluyeron familias, servicios de atención primaria y servicios privados de salud, incluso del país vecino, considerando la vulnerabilidad de las regiones fronterizas para garantizar la atención en salud. RESUMO Objetivos: conhecer o itinerário terapêutico e mostrar as implicações do câncer de mama para mulheres em município de fronteira do sul do Brasil. Métodos: pesquisa qualitativa, conduzida com base no paradigma da complexidade por meio de entrevistas com 13 mulheres com câncer de mama em tratamento ambulatorial e dados analisados pela análise temática. Resultados: os primeiros sinais e sintomas do câncer de mama foram percebidos inesperadamente, em momentos cotidianos e durante atendimentos de rotinas. Houve apoio familiar na busca por assistência, em serviços de atenção primária, mas barreiras organizacionais conduziram esse itinerário aos serviços privados de saúde, inclusive do Paraguai. O enfrentamento da doença e tratamento foi ancorado no subsistema familiar. Considerações Finais: os caminhos para o diagnóstico e tratamento do câncer de mama incluíram as famílias, os serviços de atenção primária e os serviços privados de saúde, inclusive do país vizinho, considerando a vulnerabilidade de regiões de fronteira para garantir a assistência à saúde.

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.