Functional and Structural Analysis Reveals Distinct Biological Roles of Plant Synaptotagmins in Response to Environmental Stress.

Endoplasmic reticulum-plasma membrane contact sites (ER-PM CSs) are evolutionarily conserved membrane domains found in all eukaryotes, where the ER closely interfaces with the PM. This short distance is achieved in plants through the action of tether proteins such as synaptotagmins (SYTs). Arabidopsis comprises five SYT members (SYT1-SYT5), but whether they possess overlapping or distinct biological functions remains elusive.

Peripheral membrane proteins modulate stress tolerance by safeguarding cellulose synthases.

Controlled primary cell wall remodeling allows plant growth under stressful conditions, but how these changes are conveyed to adjust cellulose synthesis is not understood. Here, we identify the TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins as new members of the cellulose synthase complex (CSC) and describe their unique and hitherto unknown dynamic association with the CSC under cellulose-deficient conditions. We find that TTLs are essential for maintaining cellulose synthesis under high-salinity conditions, establishing a stress-resilient cortical microtubule array, and stabilizing CSCs at the plasma membrane.

Phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase isoenzymes play an important role in the filling and quality of Arabidopsis thaliana seed.

Three plant-type phosphoenolpyruvate carboxylase (PPC1 to PPC3) and two phosphoenolpyruvate carboxylase kinase (PPCKs: PPCK1 and 2) genes are present in the Arabidopsis thaliana genome. In seeds, all PPC genes were found to be expressed. Examination of individual ppc mutants showed little reduction of PEPC protein and global activity, with the notable exception of PPC2 which represent the most abundant PEPC in dry seeds.

Metabolism and accumulation of hydroxylated fatty acids by castor (Ricinus comunis) seed microsomes.

Castor beans accumulate large amounts of triacylglycerols (TAGs) in the seed endosperm. This oil contains hydroxylated ricinoleic levels close to 90%, which is unique among oil seeds. The capacity to accumulate such high levels of such an unusual fatty acids is due to its specific accumulation and channeling.

The structure and flexibility analysis of the synaptotagmin 1 reveal the basis of its regulation at membrane contact sites.

Non-vesicular lipid transfer at ER and plasma membrane (PM) contact sites (CS) is crucial for the maintenance of membrane lipid homeostasis. Extended synaptotagmins (E-Syts) play a central role in this process as they act as molecular tethers of ER and PM and as lipid transfer proteins between these organelles. E-Syts are proteins constitutively anchored to the ER through an N-terminal hydrophobic segment and bind the PM via a variable number of C-terminal C2 domains.

Synaptotagmins at the endoplasmic reticulum-plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress.

Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased PM integrity under multiple abiotic stresses, such as freezing, high salt, osmotic stress, and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity.

The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis.

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization.

-Mediated Transient Gene Expression in Developing Seeds: A First Step in Making the Castor Oil Plant a Chemical Biofactory.

The castor oil plant represents a promising platform to produce oils with industrial applications. However, its use in biotechnology is limited by the absence of a well-established procedure to transform it, and a poor understanding of gene regulation and promoter use in this species. As such, a method has been developed to express proteins or hairpin-RNA in this plant, a method based on the direct injection of into the developing endosperm of castor oil fruit, enabling different constructs and promoters to be tested.

New insights in the composition of wax and sterol esters in common and mutant sunflower oils revealed by ESI-MS/MS.

Wax esters (WEs) and steryl esters (SEs) are minor components of sunflower oils formed by the esterification of long chain fatty alcohols and sterols to fatty acids. These compounds have similar carbon numbers and polarities making them difficult to separate using conventional chromatographic methods. In this study, electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) allowed the rapid and accurate profiling of WEs and SEs acyl moieties in total ester fractions of common and mutant sunflower oils with different fatty acid profiles.

Effect of level of soluble fiber and n-6/n-3 fatty acid ratio on performance of rabbit does and their litters.

The aim of this work was to study whether the dietary supplementation with soluble fiber (SF) and the reduction of the n-6/n-3 fatty acid ratio or the combination of both influences the survival, body and milk composition, and reproductive performance of rabbit does during the first four parturitions. Four diets in a 2 × 2 factorial arrangement were used with two levels of SF (7.8 vs.

Tailoring the composition of novel wax esters in the seeds of transgenic Camelina sativa through systematic metabolic engineering.

The functional characterization of wax biosynthetic enzymes in transgenic plants has opened the possibility of producing tailored wax esters (WEs) in the seeds of a suitable host crop. In this study, in addition to systematically evaluating a panel of WE biosynthetic activities, we have also modulated the acyl-CoA substrate pool, through the co-expression of acyl-ACP thioesterases, to direct the accumulation of medium-chain fatty acids. Using this combinatorial approach, we determined the additive contribution of both the varied acyl-CoA pool and biosynthetic enzyme substrate specificity to the accumulation of non-native WEs in the seeds of transgenic Camelina plants.

Field trial evaluation of the accumulation of omega-3 long chain polyunsaturated fatty acids in transgenic : Making fish oil substitutes in plants.

The global consumption of fish oils currently exceeds one million tonnes, with the natural source of these important fatty acids forming the base of marine foodwebs. Here we describe the first field-based evaluation of a terrestrial source of these essential nutrients, synthesised in the seeds of transgenic plants via the heterologous reconstitution of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway. Our data demonstrate the robust nature of this novel trait, and the feasibility of making fish oils in genetically modified crops.

Transgenic plants as a sustainable, terrestrial source of fish oils.

1An alternative, sustainable source of omega-3 long chain polyunsaturated fatty acids is widely recognized as desirable, helping to reduce pressure on current sources (wild capture fisheries) and providing a de novo source of these health beneficial fatty acids. This review will consider the efforts and progress to develop transgenic plants as terrestrial sources of omega-3 fish oils, focusing on recent developments and the possible explanations for advances in the field. We also consider the utility of such a source for use in aquaculture, since this industry is the major consumer of oceanic supplies of omega-3 fish oils.

A Transgenic Camelina sativa Seed Oil Effectively Replaces Fish Oil as a Dietary Source of Eicosapentaenoic Acid in Mice.

Background: Fish currently supplies only 40% of the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) required to allow all individuals globally to meet the minimum intake recommendation of 500 mg/d. Therefore, alternative sustainable sources are needed.

Objective: The main objective was to investigate the ability of genetically engineered Camelina sativa (20% EPA) oil (CO) to enrich tissue EPA and DHA relative to an EPA-rich fish oil (FO) in mammals.

Positional Distribution of Fatty Acids in Triacylglycerols and Phospholipids from Fillets of Atlantic Salmon (Salmo Salar) Fed Vegetable and Fish Oil Blends.

The nutritional and functional characteristics of dietary fat are related to the fatty acid (FA) composition and its positional distribution in the triacylglycerol (TAG) fraction. Atlantic salmon is an important source of healthy long chain omega 3 FA (particularly, eicosapentaenoic (EPA) and docoxahexaenoic (DHA) acids). However, the impact of lipid sources in salmon feeds on the regiospecificity of FA in the fish TAG remains to be explored.

An alternative pathway for the effective production of the omega-3 long-chain polyunsaturates EPA and ETA in transgenic oilseeds.

The synthesis and accumulation of omega-3 long-chain polyunsaturated fatty acids in transgenic Camelina sativa is demonstrated using the so-called alternative pathway. This aerobic pathway is found in a small number of taxonomically unrelated unicellular organisms and utilizes a C18 Δ9-elongase to generate C20 PUFAs. Here, we evaluated four different combinations of seed-specific transgene-derived activities to systematically determine the potential of this pathway to direct the synthesis of eicosapentaenoic acid (EPA) in transgenic plants.

Modifying the lipid content and composition of plant seeds: engineering the production of LC-PUFA.

Omega-3 fatty acids are characterized by a double bond at the third carbon atom from the end of the carbon chain. Latterly, long chain polyunsaturated omega-3 fatty acids such as eicosapentaenoic acid (EPA; 20:5Δ5,8,11,14,17) and docosahexanoic acid (DHA; 22:6 Δ4,7,10,13,16,19), which typically only enter the human diet via the consumption of oily fish, have attracted much attention. The health benefits of the omega-3 LC-PUFAs EPA and DHA are now well established.

Biochemistry of high stearic sunflower, a new source of saturated fats.

Fats based on stearic acid could be a healthier alternative to existing oils especially hydrogenated fractions of oils or palm, but only a few non-tropical species produce oils with these characteristics. In this regard, newly developed high stearic oil seed crops could be a future source of fats and hard stocks rich in stearic and oleic fatty acids. These oil crops have been obtained either by breeding and mutagenesis or by suppression of desaturases using RNA interference.

Successful high-level accumulation of fish oil omega-3 long-chain polyunsaturated fatty acids in a transgenic oilseed crop.

Omega-3 (also called n-3) long-chain polyunsaturated fatty acids (≥C20; LC-PUFAs) are of considerable interest, based on clear evidence of dietary health benefits and the concurrent decline of global sources (fish oils). Generating alternative transgenic plant sources of omega-3 LC-PUFAs, i.e.

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

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.

Reconstitution of EPA and DHA biosynthesis in arabidopsis: iterative metabolic engineering for the synthesis of n-3 LC-PUFAs in transgenic plants.

An iterative approach to optimising the accumulation of non-native long chain polyunsaturated fatty acids in transgenic plants was undertaken in Arabidopsis thaliana. The contribution of a number of different transgene enzyme activities was systematically determined, as was the contribution of endogenous fatty acid metabolism. Successive iterations were informed by lipidomic analysis of neutral, polar and acyl-CoA pools.

The modification of plant oil composition via metabolic engineering--better nutrition by design.

This article will focus on the modification of plant seed oils to enhance their nutritional composition. Such modifications will include C18 Δ6-desaturated fatty acids such as γ-linolenic and stearidonic acid, omega-6 long-chain polyunsaturated fatty acids such as arachidonic acid, as well as the omega-3 long-chain polyunsaturated fatty acids (often named 'fish oils') such as eicosapentaenoic acid and docosahexaenoic acid. We will consider how new technologies (such as synthetic biology, next-generation sequencing and lipidomics) can help speed up and direct the development of desired traits in transgenic oilseeds.

Enhancing the accumulation of omega-3 long chain polyunsaturated fatty acids in transgenic Arabidopsis thaliana via iterative metabolic engineering and genetic crossing.

The synthesis and accumulation of long chain polyunsaturated fatty acids such as eicosapentaenoic acid has previously been demonstrated in the seeds of transgenic plants. However, the obtained levels are relatively low, indicating the need for further studies and the better definition of the interplay between endogenous lipid synthesis and the non-native transgene-encoded activities. In this study we have systematically compared three different transgenic configurations of the biosynthetic pathway for eicosapentaenoic acid, using lipidomic profiling to identify metabolic bottlenecks.

Metabolic engineering of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway into transgenic plants.

Omega-3 (ω-3) very long chain polyunsaturated fatty acids (VLC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5 Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6 Δ4,7,10,13,16,19) have been shown to have significant roles in human health. Currently the primary dietary source of these fatty acids are marine fish; however, the increasing demand for fish and fish oil (in particular the expansion of the aquaculture industry) is placing enormous pressure on diminishing marine stocks. Such overfishing and concerns related to pollution in the marine environment have directed research towards the development of a viable alternative sustainable source of VLC-PUFAs.