Divergent evolution of extreme production of variant plant monounsaturated fatty acids.

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

Variation on a theme: the structures and biosynthesis of specialized fatty acid natural products in plants.

Plants are able to construct lineage-specific natural products from a wide array of their core metabolic pathways. Considerable progress has been made toward documenting and understanding, for example, phenylpropanoid natural products derived from phosphoenolpyruvate via the shikimate pathway, terpenoid compounds built using isopentyl pyrophosphate, and alkaloids generated by the extensive modification of amino acids. By comparison, natural products derived from fatty acids have received little attention, except for unusual fatty acids in seed oils and jasmonate-like oxylipins. However, scattered but numerous reports show that plants are able to generate many structurally diverse compounds from fatty acids, including some with highly elaborate and unique structural features that have novel bioproduct functionalities. Furthermore, although recent work has shed light on multiple new fatty acid natural product biosynthesis pathways and products in diverse plant species, these discoveries have not been reviewed. The aims of this work, therefore, are to (i) review and systematize our current knowledge of the structures and biosynthesis of fatty acid-derived natural products that are not seed oils or jasmonate-type oxylipins, specifically, polyacetylenic, very-long-chain, and aromatic fatty acid-derived natural products, and (ii) suggest priorities for future investigative steps that will bring our knowledge of fatty acid-derived natural products closer to the levels of knowledge that we have attained for other phytochemical classes.

Better together: Protein partnerships for lineage-specific oil accumulation.

Plant-derived oils are a major agricultural product that exist in both ubiquitous forms such as common vegetable oils and in specialized forms such as castor oil and coconut oil. These specialized oils are the result of lineage-specific metabolic pathways that create oils rich in unusual fatty acids. Considerable progress has been made toward understanding the enzymes that mediate fatty acid biosynthesis, triacylglycerol assembly, and oil storage. However, efforts to translate this knowledge into renewable bioproducts via engineered oil-producing plants and algae have had limited success. Here, we review recent evidence that protein-protein interactions in each of the three major phases of oil formation appear to have profound effects on specialized oil accumulation. We suggest that furthering our knowledge of the noncatalytic attributes of enzymes and other proteins involved in oil formation will be a critical step toward creating renewable bioproducts derived from high performing, engineered oilseeds.

Mass Spectrometry-Based Profiling of Plant Sphingolipids from Typical and Aberrant Metabolism.

Mass spectrometry has increasingly been used as a tool to complement studies of sphingolipid metabolism and biological functions in plants and other eukaryotes. Mass spectrometry is now essential for comprehensive sphingolipid analytical profiling because of the huge diversity of sphingolipid classes and molecular species in eukaryotes, particularly in plants. This structural diversity arises from large differences in polar head group glycosylation as well as carbon-chain lengths of fatty acids and desaturation and hydroxylation patterns of fatty acids and long-chain bases that together comprise the ceramide hydrophobic backbone of glycosphingolipids. The standard methods for liquid chromatography-mass spectrometry (LC-MS)-based analyses of Arabidopsis thaliana leaf sphingolipids profile >200 molecular species of four sphingolipid classes and free long-chain bases and their phosphorylated forms. While these methods have proven valuable for A. thaliana based sphingolipid research, we have recently adapted them for use with ultraperformance liquid chromatography separations of molecular species and to profile aberrant sphingolipid forms in pollen, transgenic lines, and mutants. This chapter provides updates to standard methods for LC-MS profiling of A. thaliana sphingolipids to expand the utility of mass spectrometry for plant sphingolipid research.

Production of tocotrienols in seeds of cotton (Gossypium hirsutum L.) enhances oxidative stability and offers nutraceutical potential.

Upland cotton (Gossypium hirsutum L.) is an economically important multi-purpose crop cultivated globally for fibre, seed oil and protein. Cottonseed oil also is naturally rich in vitamin E components (collectively known as tocochromanols), with α- and γ-tocopherols comprising nearly all of the vitamin E components. By contrast, cottonseeds have little or no tocotrienols, tocochromanols with a wide range of health benefits. Here, we generated transgenic cotton lines expressing the barley (Hordeum vulgare) homogentisate geranylgeranyl transferase coding sequence under the control of the Brassica napus seed-specific promoter, napin. Transgenic cottonseeds had ~twofold to threefold increases in the accumulation of total vitamin E (tocopherols + tocotrienols), with more than 60% γ-tocotrienol. Matrix assisted laser desorption ionization-mass spectrometry imaging showed that γ-tocotrienol was localized throughout the transgenic embryos. In contrast, the native tocopherols were distributed unequally in both transgenic and non-transgenic embryos. α- Tocopherol was restricted mostly to cotyledon tissues and γ-tocopherol was more enriched in the embryonic axis tissues. Production of tocotrienols in cotton embryos had no negative impact on plant performance or yield of other important seed constituents including fibre, oil and protein. Advanced generations of two transgenic events were field grown, and extracts of transgenic seeds showed increased antioxidant activity relative to extracts from non-transgenic seeds. Furthermore, refined cottonseed oil from the two transgenic events showed 30% improvement in oxidative stability relative to the non-transgenic cottonseed oil. Taken together, these materials may provide new opportunities for cottonseed co-products with enhanced vitamin E profile for improved shelf life and nutrition.

Multi-strategy engineering greatly enhances provitamin A carotenoid accumulation and stability in Arabidopsis seeds.

Staple grains with low levels of provitamin A carotenoids contribute to the global prevalence of vitamin A deficiency and therefore are the main targets for provitamin A biofortification. However, carotenoid stability during both seed maturation and postharvest storage is a serious concern for the full benefits of carotenoid biofortified grains. In this study, we utilized Arabidopsis as a model to establish carotenoid biofortification strategies in seeds. We discovered that manipulation of carotenoid biosynthetic activity by seed-specific expression of Phytoene synthase (PSY) increases both provitamin A and total carotenoid levels but the increased carotenoids are prone to degradation during seed maturation and storage, consistent with previous studies of provitamin A biofortified grains. In contrast, stacking with Orange (OR His ), a gene that initiates chromoplast biogenesis, dramatically enhances provitamin A and total carotenoid content and stability. Up to 65- and 10-fold increases of ß-carotene and total carotenoids, respectively, with provitamin A carotenoids composing over 63% were observed in the seeds containing OR His and PSY. Co-expression of Homogentisate geranylgeranyl transferase (HGGT) with OR His and PSY further increases carotenoid accumulation and stability during seed maturation and storage. Moreover, knocking-out of ß-carotene hydroxylase 2 (BCH2) by CRISPR/Cas9 not only potentially facilitates ß-carotene accumulation but also minimizes the negative effect of carotenoid over production on seed germination. Our findings provide new insights into various processes on carotenoid accumulation and stability in seeds and establish a multiplexed strategy to simultaneously target carotenoid biosynthesis, turnover, and stable storage for carotenoid biofortification in crop seeds. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-021-00046-1.

Biotechnology tools and applications for development of oilseed crops with healthy vegetable oils.

Vegetable oils, consisting principally of triacylglycerols (TAG), are major sources of calories and essential fatty acids in the human diet. The fatty acid composition of TAG is a primary determinant of the nutritional quality and health-promoting properties of vegetable oils. TAG fatty acid composition also affects the functionality and properties of vegetable oils in food applications and in food processing and preparation. Vegetable oils with improved nutritional and functional properties have been developed for oilseed crops by selection and breeding of fatty acid biosynthetic mutants. These efforts have been effective at generating vegetable oils with altered relative amounts of saturated and unsaturated fatty acids in seed TAG, but are constrained by insufficient genetic diversity for producing oils with "healthy" fatty acids that are not typically found in major oilseeds. The development and application of biotechnological tools have instead enabled the generation of oilseeds that produce novel fatty acid compositions with improved nutritional value by the introduction of genes from alternative sources, including plants, bacteria, and fungi. These tools have also allowed the generation of desired oil compositions that have proven difficult to obtain by breeding without compromised performance in selected oilseed crops. Here, we review biotechnological tools for increasing crop genetic diversity and their application for commercial or proof-of-principal development of oilseeds with expanded utility for food and feed applications and higher value nutritional and nutraceutical markets.

Stearidonic-Enriched Soybean Oil Modulates Obesity, Glucose Metabolism, and Fatty Acid Profiles Independently of Akkermansia muciniphila.

SCOPE: Previous studies have suggested that diets rich in omega-3 and low in omega-6 long-chain polyunsaturated fatty acids (PUFAs) can limit the development of metabolic syndrome (MetS). Transgenic soybeans yielding oils enriched for omega-3 PUFAs represent a new and readily-available option for incorporating omega-3 PUFAs into diets to provide health benefits. METHODS AND RESULTS: Transgenic soybean oils, enriched for either stearidonic acid (SDA) or eicosapentaenoic acid (EPA), are incorporated into diets to test their effects on limiting the development of MetS in a mouse model of diet-induced obesity. Supplementation with SDA- but not EPA-enriched oils improved features of MetS compared to feeding a control wild-type oil. Because previous studies have linked the gut microorganism Akkermansia muciniphila to the metabolic effects of feeding omega-3 PUFAs, the causal contribution of A. muciniphila to mediating the metabolic benefits provided by SDA-enriched diets is investigated. Although A. muciniphila is not required for SDA-induced metabolic improvements, this microorganism does modulate levels of saturated and mono-unsaturated fatty acids in host adipose tissues. CONCLUSION: Together, these findings support the utilization of SDA-enriched diets to modulate weight gain, glucose metabolism, and fatty acid profiles of liver and adipose tissue.

Unregulated Sphingolipid Biosynthesis in Gene-Edited Arabidopsis ORM Mutants Results in Nonviable Seeds with Strongly Reduced Oil Content.

Orosomucoid-like proteins (ORMs) interact with serine palmitoyltransferase (SPT) to negatively regulate sphingolipid biosynthesis, a reversible process critical for balancing the intracellular sphingolipid levels needed for growth and programmed cell death. Here, we show that ORM1 and ORM2 are essential for life cycle completion in Arabidopsis (Arabidopsis thaliana). Seeds from orm1 -/- orm2 -/- mutants, generated by crossing CRISPR/Cas9 knockout mutants for each gene, accumulated high levels of ceramide, indicative of unregulated sphingolipid biosynthesis. orm1 -/- orm2 -/- seeds were nonviable, displayed aberrant embryo development, and had >80% reduced oil content versus wild-type seeds. This phenotype was mimicked in Arabidopsis seeds expressing the SPT subunit LCB1 lacking its first transmembrane domain, which is critical for ORM-mediated regulation of SPT. We identified a mutant for ORM1 lacking one amino acid (Met-51) near its second transmembrane domain that retained its membrane topology. Expressing this allele in the orm2 background yielded plants that did not advance beyond the seedling stage, hyperaccumulated ceramides, and showed altered organellar structures and increased senescence- and pathogenesis-related gene expression. These seedlings also showed upregulated expression of genes for sphingolipid catabolic enzymes, pointing to additional mechanisms for maintaining sphingolipid homeostasis. ORM1 lacking Met-51 had strongly impaired interactions with LCB1 in a yeast (Saccharomyces cerevisiae) model, providing structural clues about regulatory interactions between ORM and SPT.

Plant unusual fatty acids: learning from the less common.

The plant kingdom contains an abundance of structurally diverse fatty acids referred to as unusual fatty acids. Unusual fatty acids on plant surfaces can form polyesters that contribute to the function of cutin as a barrier for water loss and pathogen protection. Unusual fatty acids are also found as abundant components of seed oils of selected species and often confer desirable properties for industrial and nutritional applications. Here, we review recent findings on the biosynthesis and metabolism of unusual fatty acids in cutin and seed oils and use of this information for enzyme structure-function studies and seed oil metabolic engineering. We also highlight the recent discovery of unusual fatty acids that are formed from a previously undescribed variation of fatty acid elongation.

Metabolic engineering of soybean seeds for enhanced vitamin E tocochromanol content and effects on oil antioxidant properties in polyunsaturated fatty acid-rich germplasm.

Soybean seeds produce oil enriched in oxidatively unstable polyunsaturated fatty acids (PUFAs) and are also a potential biotechnological platform for synthesis of oils with nutritional omega-3 PUFAs. In this study, we engineered soybeans for seed-specific expression of a barley homogentisate geranylgeranyl transferase (HGGT) transgene alone and with a soybean γ-tocopherol methyltransferase (γ-TMT) transgene. Seeds for HGGT-expressing lines had 8- to 10-fold increases in total vitamin E tocochromanols, principally as tocotrienols, with little effect on seed oil or protein concentrations. Tocochromanols were primarily in δ- and γ-forms, which were shifted largely to α- and ß-tocochromanols with γ-TMT co-expression. We tested whether oxidative stability of conventional or PUFA-enhanced soybean oil could be improved by metabolic engineering for increased vitamin E antioxidants. Selected lines were crossed with a stearidonic acid (SDA, 18:4Δ6,9,12,15)-producing line, resulting in progeny with oil enriched in SDA and α- or γ-linoleic acid (ALA, 18:3Δ9,12,15 or GLA, 18:3Δ6,9,12), from transgene segregation. Oil extracted from HGGT-expressing lines had ≥6-fold increase in free radical scavenging activity compared to controls. However, the oxidative stability index of oil from vitamin E-enhanced lines was ~15% lower than that of oil from non-engineered seeds and nearly the same or modestly increased in oil from the GLA, ALA and SDA backgrounds relative to controls. These findings show that soybean is an effective platform for producing high levels of free-radical scavenging vitamin E antioxidants, but this trait may have negative effects on oxidative stability of conventional oil or only modest improvement of the oxidative stability of PUFA-enhanced oil.

Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop.

Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run-off on otherwise barren farmland. We demonstrate that pennycress can serve as a user-friendly model system akin to Arabidopsis that is well-suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring-type Spring 32-10 inbred line (1C DNA content of 539 Mb; 2n = 14), identifying variation that may explain phenotypic differences with winter-type pennycress, as well as predominantly a one-to-one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium-mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR-Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production and creating an edible seed oil comparable to that of canola. We also stably transformed pennycress with the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene, producing low-viscosity acetyl-triacylglycerol-containing seed oil suitable as a diesel-engine drop-in fuel. Adoption of pennycress as a model system will accelerate oilseed-crop translational research and facilitate pennycress' rapid domestication to meet the growing sustainable food and fuel demands.

Expression of the Arabidopsis WRINKLED 1 transcription factor leads to higher accumulation of palmitate in soybean seed.

Soybean (Glycine max [L.] Merr.) is a commodity crop highly valued for its protein and oil content. The high percentage of polyunsaturated fatty acids in soybean oil results in low oxidative stability, which is a key parameter for usage in baking, high temperature frying applications, and affects shelf life of packaged products containing soybean oil. Introduction of a seed-specific expression cassette carrying the Arabidopsis transcription factor WRINKLED1 (AtWRI1) into soybean, led to seed oil with levels of palmitate up to approximately 20%. Stacking of the AtWRI1 transgenic allele with a transgenic locus harbouring the mangosteen steroyl-ACP thioesterase (GmFatA) resulted in oil with total saturates up to 30%. The creation of a triple stack in soybean, wherein the AtWRI1 and GmFatA alleles were combined with a FAD2-1 silencing allele led to the synthesis of an oil with 28% saturates and approximately 60% oleate. Constructs were then assembled that carry a dual FAD2-1 silencing element/GmFatA expression cassette, alone or combined with an AtWRI1 cassette. These plasmids are designated pPTN1289 and pPTN1301, respectively. Transgenic events carrying the T-DNA of pPTN1289 displayed an oil with stearate levels between 18% and 25%, and oleate in the upper 60%, with reduced palmitate (<5%). While soybean events harboring transgenic alleles of pPTN1301 had similar levels of stearic and oleate levels as that of the pPTRN1289 events, but with levels of palmitate closer to wild type. The modified fatty acid composition results in an oil with higher oxidative stability, and functionality attributes for end use in baking applications.

Discontinuous fatty acid elongation yields hydroxylated seed oil with improved function.

The biosynthesis of 'unusual' fatty acids with structures that deviate from the common C16 and C18 fatty acids has evolved numerous times in the plant kingdom. Characterization of unusual fatty acid biosynthesis has enabled increased understanding of enzyme substrate properties, metabolic plasticity and oil functionality. Here, we report the identification of a novel pathway for hydroxy fatty acid biosynthesis based on the serendipitous discovery of two C24 fatty acids containing hydroxyl groups at the 7 and 18 carbon atoms as major components of the seed oil of Orychophragmus violaceus, a China-native Brassicaceae. Biochemical and genetic evidence are presented for premature or 'discontinuous' elongation of a 3-OH intermediate by a divergent 3-ketoacyl-CoA (coenzyme A) synthase during a chain extension cycle as the origin of the 7-OH group of the dihydroxy fatty acids. Tribology studies revealed superior high-temperature lubricant properties for O. violaceus seed oil compared to castor oil, a high-performance vegetable oil lubricant. These findings provide a direct pathway for designing a new class of environmentally friendly lubricants and unveil the potential of O. violaceus as a new industrial oilseed crop.

Towards the synthetic design of camelina oil enriched in tailored acetyl-triacylglycerols with medium-chain fatty acids.

The ability to manipulate expression of key biosynthetic enzymes has allowed the development of genetically modified plants that synthesise unusual lipids that are useful for biofuel and industrial applications. By taking advantage of the unique activities of enzymes from different species, tailored lipids with a targeted structure can be conceived. In this study we demonstrate the successful implementation of such an approach by metabolically engineering the oilseed crop Camelina sativa to produce 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) with medium-chain fatty acids (MCFAs). Different transgenic camelina lines that had been genetically modified to produce MCFAs through the expression of MCFA-specific thioesterases and acyltransferases were retransformed with the Euonymus alatus gene for diacylglycerol acetyltransferase (EaDAcT) that synthesises acetyl-TAGs. Concomitant RNAi suppression of acyl-CoA:diacylglycerol acyltransferase increased the levels of acetyl-TAG, with up to 77 mole percent in the best lines. However, the total oil content was reduced. Analysis of the composition of the acetyl-TAG molecular species using electrospray ionisation mass spectrometry demonstrated the successful synthesis of acetyl-TAG containing MCFAs. Field growth of high-yielding plants generated enough oil for quantification of viscosity. As part of an ongoing design-test-learn cycle, these results, which include not only the synthesis of 'designer' lipids but also their functional analysis, will lead to the future production of such molecules tailored for specific applications.

GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) Is a GlcNAc-Containing Glycosylinositol Phosphorylceramide Glycosyltransferase.

Glycosylinositol phosphorylceramides (GIPCs), which have a ceramide core linked to a glycan headgroup of varying structures, are the major sphingolipids in the plant plasma membrane. Recently, we identified the major biosynthetic genes for GIPC glycosylation in Arabidopsis (Arabidopsis thaliana) and demonstrated that the glycan headgroup is essential for plant viability. However, the function of GIPCs and the significance of their structural variation are poorly understood. Here, we characterized the Arabidopsis glycosyltransferase GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) and showed that it is responsible for the glycosylation of a subgroup of GIPCs found in seeds and pollen that contain GlcNAc and GlcN [collectively GlcN(Ac)]. In Arabidopsis gint1 plants, loss of the GlcN(Ac) GIPCs did not affect vegetative growth, although seed germination was less sensitive to abiotic stress than in wild-type plants. However, in rice, where GlcN(Ac) containing GIPCs are the major GIPC subgroup in vegetative tissue, loss of GINT1 was seedling lethal. Furthermore, we could produce, de novo, "rice-like" GlcN(Ac) GIPCs in Arabidopsis leaves, which allowed us to test the function of different sugars in the GIPC headgroup. This study describes a monocot GIPC biosynthetic enzyme and shows that its Arabidopsis homolog has the same biochemical function. We also identify a possible role for GIPCs in maintaining cell-cell adhesion.

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

Modified fatty acids (mFA) have diverse uses; for example, 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. Thus, 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 co-expression 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. 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.

Provitamin A biofortification of cassava enhances shelf life but reduces dry matter content of storage roots due to altered carbon partitioning into starch.

Storage roots of cassava (Manihot esculenta Crantz), a major subsistence crop of sub-Saharan Africa, are calorie rich but deficient in essential micronutrients, including provitamin A ß-carotene. In this study, ß-carotene concentrations in cassava storage roots were enhanced by co-expression of transgenes for deoxy-d-xylulose-5-phosphate synthase (DXS) and bacterial phytoene synthase (crtB), mediated by the patatin-type 1 promoter. Storage roots harvested from field-grown plants accumulated carotenoids to ≤50 µg/g DW, 15- to 20-fold increases relative to roots from nontransgenic plants. Approximately 85%-90% of these carotenoids accumulated as all-trans-ß-carotene, the most nutritionally efficacious carotenoid. ß-Carotene-accumulating storage roots displayed delayed onset of postharvest physiological deterioration, a major constraint limiting utilization of cassava products. Large metabolite changes were detected in ß-carotene-enhanced storage roots. Most significantly, an inverse correlation was observed between ß-carotene and dry matter content, with reductions of 50%-60% of dry matter content in the highest carotenoid-accumulating storage roots of different cultivars. Further analysis confirmed a concomitant reduction in starch content and increased levels of total fatty acids, triacylglycerols, soluble sugars and abscisic acid. Potato engineered to co-express DXS and crtB displayed a similar correlation between ß-carotene accumulation, reduced dry matter and starch content and elevated oil and soluble sugars in tubers. Transcriptome analyses revealed a reduced expression of genes involved in starch biosynthesis including ADP-glucose pyrophosphorylase genes in transgenic, carotene-accumulating cassava roots relative to nontransgenic roots. These findings highlight unintended metabolic consequences of provitamin A biofortification of starch-rich organs and point to strategies for redirecting metabolic flux to restore starch production.

A Specialized Diacylglycerol Acyltransferase Contributes to the Extreme Medium-Chain Fatty Acid Content of Cuphea Seed Oil.

Seed oils of many Cuphea sp. contain >90% of medium-chain fatty acids, such as decanoic acid (10:0). These seed oils, which are among the most compositionally variant in the plant kingdom, arise from specialized fatty acid biosynthetic enzymes and specialized acyltransferases. These include lysophosphatidic acid acyltransferases (LPAT) and diacylglycerol acyltransferases (DGAT) that are required for successive acylation of medium-chain fatty acids in the sn-2 and sn-3 positions of seed triacylglycerols (TAGs). Here we report the identification of a cDNA for a DGAT1-type enzyme, designated CpuDGAT1, from the transcriptome of C. avigera var pulcherrima developing seeds. Microsomes of camelina (Camelina sativa) seeds engineered for CpuDGAT1 expression displayed DGAT activity with 10:0-CoA and the diacylglycerol didecanoyl, that was approximately 4-fold higher than that in camelina seed microsomes lacking CpuDGAT1. In addition, coexpression in camelina seeds of CpuDGAT1 with a C. viscosissima FatB thioesterase (CvFatB1) that generates 10:0 resulted in TAGs with nearly 15 mol % of 10:0. More strikingly, expression of CpuDGAT1 and CvFatB1 with the previously described CvLPAT2, a 10:0-CoA-specific Cuphea LPAT, increased 10:0 amounts to 25 mol % in camelina seed TAG. These TAGs contained up to 40 mol % 10:0 in the sn-2 position, nearly double the amounts obtained from coexpression of CvFatB1 and CvLPAT2 alone. Although enriched in diacylglycerol, 10:0 was not detected in phosphatidylcholine in these seeds. These findings are consistent with channeling of 10:0 into TAG through the combined activities of specialized LPAT and DGAT activities and demonstrate the biotechnological use of these enzymes to generate 10:0-rich seed oils.

Effect of Extraction Method on the Oxidative Stability of Camelina Seed Oil Studied by Differential Scanning Calorimetry.

Camelina seed is a new alternative omega-3 source attracting growing interest. However, it is susceptible to oxidation due to its high omega-3 content. The objective of this study was to improve the oxidative stability of the camelina seed oil at the extraction stage in order to eliminate or minimize the use of additive antioxidants. Camelina seed oil extracts were enriched in terms of natural antioxidants using ethanol-modified supercritical carbon dioxide (SC-CO2 ) extraction. Oxidative stability of the camelina seed oils extracted by ethanol modified SC-CO2 was studied by differential scanning calorimeter (DSC), and compared with cold press, hexane, and SC-CO2 methods. Nonisothermal oxidation kinetics of the oils obtained by different extraction methods were studied by DSC at varying heating rates (2.5, 5, 10, and 15 °C/min). Increasing ethanol level in the ethanol-modified SC-CO2 increased the oxidative stability. Based on oxidation onset temperatures (Ton ), SC-CO2 containing 10% ethanol yielded the most stable oil. Oxidative stability depended on the type and content of the polar fractions, namely, phenolic compounds and phospholipids. Phenolic compounds acted as natural antioxidants, whereas increased phospholipid contents decreased the stability. Study has shown that the oxidative stability of the oils can be improved at the extraction stage and this may eliminate the need for additive antioxidants.