Two Acyltransferases Contribute Differently to Linolenic Acid Levels in Seed Oil.

Acyltransferases are key contributors to triacylglycerol (TAG) synthesis and, thus, are of great importance for seed oil quality. The effects of increased or decreased expression of ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) on seed lipid composition were assessed in several Camelina sativa lines. Furthermore, in vitro assays of acyltransferases in microsomal fractions prepared from developing seeds of some of these lines were performed. Decreased expression of DGAT1 led to an increased percentage of 18:3n-3 without any change in total lipid content of the seed. The tri-18:3 TAG increase occurred predominantly in the cotyledon, as determined with matrix-assisted laser desorption/ionization-mass spectrometry, whereas species with two 18:3n-3 acyl groups were elevated in both cotyledon and embryonal axis. PDAT overexpression led to a relative increase of 18:2n-6 at the expense of 18:3n-3, also without affecting the total lipid content. Differential distributions of TAG species also were observed in different parts of the seed. The microsomal assays revealed that C.sativa seeds have very high activity of diacylglycerol-phosphatidylcholine interconversion. The combination of analytical and biochemical data suggests that the higher 18:2n-6 content in the seed oil of the PDAT overexpressors is due to the channeling of fatty acids from phosphatidylcholine into TAG before being desaturated to 18:3n-3, caused by the high activity of PDAT in general and by PDAT specificity for 18:2n-6. The higher levels of 18:3n-3 in DGAT1-silencing lines are likely due to the compensatory activity of a TAG-synthesizing enzyme with specificity for this acyl group and more desaturation of acyl groups occurring on phosphatidylcholine.

Transcriptional stimulation of rate-limiting components of the autophagic pathway improves plant fitness.

Autophagy is a major catabolic process whereby autophagosomes deliver cytoplasmic content to the lytic compartment for recycling. Autophagosome formation requires two ubiquitin-like systems conjugating Atg12 with Atg5, and Atg8 with lipid phosphatidylethanolamine (PE), respectively. Genetic suppression of these systems causes autophagy-deficient phenotypes with reduced fitness and longevity. We show that Atg5 and the E1-like enzyme, Atg7, are rate-limiting components of Atg8-PE conjugation in Arabidopsis. Overexpression of ATG5 or ATG7 stimulates Atg8 lipidation, autophagosome formation, and autophagic flux. It also induces transcriptional changes opposite to those observed in atg5 and atg7 mutants, favoring stress resistance and growth. As a result, ATG5- or ATG7-overexpressing plants exhibit increased resistance to necrotrophic pathogens and oxidative stress, delayed aging and enhanced growth, seed set, and seed oil content. This work provides an experimental paradigm and mechanistic insight into genetic stimulation of autophagy in planta and shows its efficiency for improving plant productivity.

Phenotypic characterization and candidate gene analysis of a short kernel and brassinosteroid insensitive mutant from hexaploid oat (Avena sativa).

In an ethyl methanesulfonate oat (Avena sativa) mutant population we have found a mutant with striking differences to the wild-type (WT) cv. Belinda. We phenotyped the mutant and compared it to the WT. The mutant was crossed to the WT and mapping-by-sequencing was performed on a pool of F2 individuals sharing the mutant phenotype, and variants were called. The impacts of the variants on genes present in the reference genome annotation were estimated. The mutant allele frequency distribution was combined with expression data to identify which among the affected genes was likely to cause the observed phenotype. A brassinosteroid sensitivity assay was performed to validate one of the identified candidates. A literature search was performed to identify homologs of genes known to be involved in seed shape from other species. The mutant had short kernels, compact spikelets, altered plant architecture, and was found to be insensitive to brassinosteroids when compared to the WT. The segregation of WT and mutant phenotypes in the F2 population was indicative of a recessive mutation of a single locus. The causal mutation was found to be one of 123 single-nucleotide polymorphisms (SNPs) spanning the entire chromosome 3A, with further filtering narrowing this down to six candidate genes. In-depth analysis of these candidate genes and the brassinosteroid sensitivity assay suggest that a Pro303Leu substitution in AVESA.00010b.r2.3AG0419820.1 could be the causal mutation of the short kernel mutant phenotype. We identified 298 oat proteins belonging to orthogroups of previously published seed shape genes, with AVESA.00010b.r2.3AG0419820.1 being the only of these affected by a SNP in the mutant. The AVESA.00010b.r2.3AG0419820.1 candidate is functionally annotated as a GSK3/SHAGGY-like kinase with homologs in Arabidopsis, wheat, barley, rice, and maize, with several of these proteins having known mutants giving rise to brassinosteroid insensitivity and shorter seeds. The substitution in AVESA.00010b.r2.3AG0419820.1 affects a residue with a known gain-of function substitution in Arabidopsis BRASSINOSTEROID-INSENSITIVE2. We propose a gain-of-function mutation in AVESA.00010b.r2.3AG0419820.1 as the most likely cause of the observed phenotype, and name the gene AsGSK2.1. The findings presented here provide potential targets for oat breeders, and a step on the way towards understanding brassinosteroid signaling, seed shape and nutrition in oats.

Acyltransferases Regulate Oil Quality in Camelina sativa Through Both Acyl Donor and Acyl Acceptor Specificities.

Camelina sativa is an emerging biotechnology oil crop. However, more information is needed regarding its innate lipid enzyme specificities. We have therefore characterized several triacylglycerol (TAG) producing enzymes by measuring in vitro substrate specificities using different combinations of acyl-acceptors (diacylglycerol, DAG) and donors. Specifically, C. sativa acyl-CoA:diacylglycerol acyltransferase (DGAT) 1 and 2 (which both use acyl-CoA as acyl donor) and phospholipid:diacylglycerol acyltransferase (PDAT, with phosphatidylcoline as acyl donor) were studied. The results show that the DGAT1 and DGAT2 specificities are complementary, with DGAT2 exhibiting a high specificity for acyl acceptors containing only polyunsaturated fatty acids (FAs), whereas DGAT1 prefers acyl donors with saturated and monounsaturated FAs. Furthermore, the combination of substrates that resulted in the highest activity for DGAT2, but very low activity for DGAT1, corresponds to TAG species previously shown to increase in C. sativa seeds with downregulated DGAT1. Similarly, the combinations of substrates that gave the highest PDAT1 activity were also those that produce the two TAG species (54:7 and 54:8 TAG) with the highest increase in PDAT overexpressing C. sativa seeds. Thus, the in vitro data correlate well with the changes in the overall fatty acid profile and TAG species in C. sativa seeds with altered DGAT1 and PDAT activity. Additionally, in vitro studies of C. sativa phosphatidycholine:diacylglycerol cholinephosphotransferase (PDCT), another activity involved in TAG biosynthesis, revealed that PDCT accepts substrates with different desaturation levels. Furthermore, PDCT was unable to use DAG with ricineoleyl groups, and the presence of this substrate also inhibited PDCT from using other DAG-moieties. This gives insights relating to previous in vivo studies regarding this enzyme.

Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts.

BACKGROUND: Photosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants and can be grown on non-arable land. Among many microalgae considered for biofuel production, Nannochloropsis oceanica (CCMP1779) is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). The committed step in TAG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). Remarkably, a total of 13 putative DGAT-encoding genes have been previously identified in CCMP1779 but most have not yet been studied in detail. RESULTS: Based on their expression profile, six out of 12 type-2 DGAT-encoding genes (NoDGTT1-NoDGTT6) were chosen for their possible role in TAG biosynthesis and the respective cDNAs were expressed in a TAG synthesis-deficient mutant of yeast. Yeast expressing NoDGTT5 accumulated TAG to the highest level. Over-expression of NoDGTT5 in CCMP1779 grown in N-replete medium resulted in levels of TAG normally observed only after N deprivation. Reduced growth rates accompanied NoDGTT5 over-expression in CCMP1779. Constitutive expression of NoDGTT5 in Arabidopsis thaliana was accompanied by increased TAG content in seeds and leaves. A broad substrate specificity for NoDGTT5 was revealed, with preference for unsaturated acyl groups. Furthermore, NoDGTT5 was able to successfully rescue the Arabidopsis tag1-1 mutant by restoring the TAG content in seeds. CONCLUSIONS: Taken together, our results identified NoDGTT5 as the most promising gene for the engineering of TAG synthesis in multiple hosts among the 13 DGAT-encoding genes of N. oceanica CCMP1779. Consequently, this study demonstrates the potential of NoDGTT5 as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production.

Tuning the pH-shift protein-isolation method for maximum hemoglobin-removal from blood rich fish muscle.

A main challenge preventing optimal use of protein isolated from unconventional raw materials (e.g., small pelagic fish and fish by-products) using the pH-shift method is the difficulty to remove enough heme-pigments. Here, the distribution of hemoglobin (Hb) in the different fractions formed during pH-shift processing was studied using Hb-fortified cod mince. Process modifications, additives and prewashing were then investigated to further facilitate Hb-removal. The alkaline pH-shift process version could remove considerably more Hb (77%) compared to the acidic version (37%) when proteins were precipitated at pH 5.5; most Hb was removed during dewatering. Protein precipitation at pH 6.5 improved total Hb removal up to 91% and 74% during alkaline and acid processing, respectively. Adding phytic acid to the first supernatant of the alkaline process version yielded 93% Hb removal. Combining one prewash with phytic acid at pH 5.5 followed by alkaline/acid pH-shift processing increased Hb removal up to 96/92%.

Effect of alkaline pH-shift processing on in vitro gastrointestinal digestion of herring (Clupea harengus) fillets.

The effect of alkaline pH-shift processing on herring (Clupea harengus) protein oxidation, salt solubility and digestibility, has been evaluated. For the latter, herring mince and pH-shift produced herring protein isolate, both raw and heat-treated, were digested using a static gastrointestinal in vitro model. The pH-shift process resulted in drastically lowered protein salt solubility and increased lipid oxidation while protein carbonyl formation was unaffected. Yet, no significant differences in the degree of hydrolysis (DH) were observed between mince and isolates after completed gastrointestinal digestion, something which was confirmed by a similar release of proteinaceous material <3 kDa and similar free amino acid profiles. The polypeptide profiles of digested samples however revealed that two peptides (33 and 36 kDa) were present in larger amounts in the digested protein isolate compared to the digested herring mince. The results indicate that alkaline pH-shift processing had limited quantitative influence on the gastrointestinal digestibility of herring proteins despite its negative effects on protein salt solubility and lipid oxidation.

Changes in salt solubility and microstructure of proteins from herring (Clupea harengus) after pH-shift processing.

Salt solubility of pH-shift isolated herring (Clupea harengus) muscle proteins was studied in relation to pH exposure and microstructure using transmission electron microscopy (TEM). Using protein solubilization at pH 11.2 with subsequent precipitation at pH 5.5, salt solubility of the proteins decreased from 78 to 17%. By precipitating the alkali-solubilized proteins at the pH of native herring muscle, 6.5, salt solubility only decreased to 59%, proving that pH values between 6.5 and 5.5 affected protein salt solubility more than the pH cycle 6.5 → 11.2 → 6.5. Precipitation at pH 5.5 resulted in hydrogen bonds, hydrophobic interactions, and S-S bridges, whereas precipitation at pH 6.5 resulted only in the formation of hydrophobic interactions. The alkaline pH-shift isolation process severely rearranged the protein microstructure, with precipitation at pH 6.5 forming a finer, more homogeneous network than precipitation at pH 5.5. The former protein isolate also contained less lipid oxidation products and formed more deformable gels, without affecting protein yield.

Dietary herring improves plasma lipid profiles and reduces atherosclerosis in obese low-density lipoprotein receptor-deficient mice.

Diet is a significant modifiable risk factor for cardiovascular disease and high fish intake has been associated with vascular health in population studies. However, intervention studies have been inconclusive. In this study, male low-density lipoprotein receptor-deficient mice were given 16-week high fat/high sucrose diets, supplemented with either minced herring fillets or minced beef. The diets were matched in total fat and cholesterol content; taurine content and fatty acid composition was analysed. Body weights were recorded throughout the study; plasma lipids were analysed at week 8 and 16. Body composition and adipocyte size were evaluated at study end. Atherosclerosis was evaluated at week 12 (ultrasound) and at termination (en face histology). Herring-fed mice had a higher proportion of long-chain n-3 polyunsaturated fatty acids in the hepatic triacylglycerides (TAG) and phospholipid fractions. The herring-fed mice had increased body weight (P=0.007), and reduced epididymal adipocyte size (P=0.009), despite similar food intake and body composition as the beef-fed mice. The herring-fed mice had lower plasma TAG and very-low-density lipoprotein (VLDL)-cholesterol concentrations throughout the study (TAG; P=0.0012 and 0.004, VLDL-cholesterol; P=0.006 and 0.041, week 8 and 16, respectively). At week 16, the herring-fed had higher plasma concentrations of HDL-cholesterol (P=0.004) and less atherosclerotic lesions in the aortic arch (P=0.007) compared with the beef-fed mice. In conclusion, dietary herring in comparison to beef markedly improved vascular health in this mouse model, suggesting that herring provides an added value beyond its content of macronutrients.

Protein isolation from gutted herring (Clupea harengus) using pH-shift processes.

Herring ( Clupea harengus ) and other pelagic fish species are mainly used for fish meal and oil production and not for human consumption. In this study, acid pH-shift processing and alkaline pH-shift processing were used to isolate proteins from whole gutted herring with the aim to investigate the potential use of herring proteins as a food ingredient. The acid and alkaline processes gave rise to similar protein yields, 59.3 and 57.3%. The protein isolates from both processes had a significantly (p < 0.05) whiter color and higher protein and lower lipid contents than the starting material. The removal of ash was >80% for both processes, with a trend (p = 0.07) toward higher removal during the alkaline process. Also, Ca and Mg removal was significantly (p < 0.05) higher during the alkaline process. The isolated proteins from the acid process contained myosin degradation products and had a lower salt solubility than proteins from the alkaline process. Both protein isolates had an amino acid profile meeting the recommendations for adults according to FAO/WHO/UNU and could produce a surimi gel of medium strength. The results show that pH-shift processing could be a valuable method for the production of functional food proteins from gutted herring.

Removal of lipids, dioxins, and polychlorinated biphenyls during production of protein isolates from baltic herring ( Clupea harengus ) using pH-shift processes.

Dioxins and PCBs are toxic, lipophilic, and persistent substances that impose a serious health threat. A major risk of exposure to these toxic substances is consumption of fish from polluted waters, such as the Baltic Sea. The aim of this study was to investigate if pH-shift processing of Baltic herring with elevated toxicity levels could be used to produce a protein isolate with low fat content and, thereby, reduced dioxin and PCB levels. Both acid (pH 2.7) and alkaline (pH 11.2) pH-shift processing were investigated and resulted in efficient reduction of fat, dioxin, and PCB levels. A reduction of 70-80% per amount of protein was determined for all of these parameters. The amounts, and thus the removal, of lipids and dioxins (R(2) = 0.952) as well as lipids and PCBs (R(2) = 0.996) were highly correlated (p < 0.01). A mass balance of the alkaline pH-shift process showed that most of the fat and pollutants were found in the floating fat emulsion layer of the first centrifugation, followed by the pellet of the first centrifugation. These data show that the pH-shift protein isolation technique can be used to process herring with elevated dioxin and PCB levels and thereby increase the usage possibilities of such fish.