Magel2, a Prader-Willi syndrome candidate gene, modulates the activities of circadian rhythm proteins in cultured cells

<p>Abstract</p> <p>Background</p> <p>The Magel2 gene is most highly expressed in the suprachiasmatic nucleus of the hypothalamus, where its expression cycles in a circadian pattern comparable to that of clock-controlled genes. Mice lacking the <it>Magel2 </it>gene have hypothalamic dysfunction, including circadian defects that include reduced and fragmented total activity, excessive activity during the subjective day, but they have a normal circadian period. Magel2 is a member of the MAGE family of proteins that have various roles in cellular function, but the specific function of Magel2 is unknown.</p> <p>Methods</p> <p>We used a variety of cell-based assays to determine whether Magel2 modifies the properties of core circadian rhythm proteins.</p> <p>Results</p> <p>Magel2 represses the activity of the Clock:Bmal1 heterodimer in a Per2-luciferase assay. Magel2 interacts with Bmal1 and with Per2 as measured by co-immunoprecipitation in co-transfected cells, and exhibits a subcellular distribution consistent with these interactions when visualized by immunofluorescence. As well, Magel2 induces the redistribution of the subcellular localization of Clock towards the cytoplasm, in contrast to the nucleus-directed effect of Bmal1 on Clock subcellular localization.</p> <p>Conclusion</p> <p>Consistent with the blunted circadian rhythm observed in <it>Magel2-</it>null mice, these data suggest that Magel2 normally promotes negative feedback regulation of the cellular circadian cycle, through interactions with key core circadian rhythm proteins.</p

Down‐regulation of key genes involved in carbon metabolism in Medicago truncatula results in increased lipid accumulation in vegetative tissue

Alfalfa (Medicago sativa L.), is the most widely grown perennial forage crop, which is a close relative of the model diploid legume Medicago truncatula. However, use of alfalfa lead to substantial greenhouse gas emissions and economic losses related to inefficiencies in rumen fermentation. The provision of supplemental lipids has been used as a strategy to mitigate these issues, but it is a costly approach. The ability to enhance lipid content within the vegetative tissues of alfalfa would therefore be very advantageous. As such, our aim was to assess and select gene candidates to increase total shoot lipid content in M. truncatula using a virus‐induced gene silencing (VIGS) approach. We targeted gene homologs of the SUGAR‐DEPENDANT 1 (SDP1), ADP‐GLUCOSE‐PYROPHOSPHORYLASE SMALL SUBUNIT 1 (APS1), TRIGALACTOSYLDIACYLGLYCEROL 5 (TGD5) and PEROXISOMAL ABC TRANSPORTER 1 (PXA1) in M. truncatula for silencing. Reduced target transcript levels were confirmed and changes of shoot lipid content and fatty acid composition were measured. Silencing of SDP1, APS1 and PXA1 each resulted in significant increases in shoot total lipid content. Significantly increased proportions of α‐linolenic acid (18:3Δ9cis,12cis,15cis) were observed and stearic acid (18:0) levels significantly decreased in the total acyl lipids extracted from vegetative tissues of each of the M. truncatula silenced plants. In contrast, palmitic acid (16:0) levels were significantly decreased in only SDP1 and PXA1‐silenced plants. Genes of PXA1 and SDP1 would be ideal targets for mutation as a means of improving the quality of alfalfa for increasing feed efficiency and minimizing greenhouse gas emissions from livestock production in the future

Metabolic control analysis of triacylglycerol accumulation in oilseed rape

The increasing global demand for vegetable oils will only be met if there are significant improvements in the productivity of the major oil crops, such as oilseed rape. Metabolic engineering offers the prospect of further gains in yield beyond that already achieved by breeding and selection but requires guidance as to the changes that need to be made. Metabolic Control Analysis, through measurement and estimation of flux control coefficients, can indicate which enzymes have the most influence on a desired flux. Some experiments have previously reported flux control coefficients for oil accumulation in the seeds of oilseed rape, and others have measured control coefficient distributions for multi-enzyme segments of oil synthesis in seed embryo metabolism measured in vitro. In addition, other reported manipulations of oil accumulation contain results that are exploited further here to calculate previously unknown flux control coefficients. These results are then assembled within a framework that allows an integrated interpretation of the controls on oil accumulation from the assimilation of CO2 to deposition of oil in the seed. The analysis shows that the control is distributed to an extent that the gains from amplifying any single target are necessarily limited, but there are candidates for joint amplification that are likely to act synergistically to produce much more significant gains

Functional Characterization of Lysophosphatidylcholine: Acyl-CoA Acyltransferase Genes From Sunflower (Helianthus annuus L.)

Lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23) is an evolutionarily conserved key enzyme in the Lands cycle that catalyzes acylation of lysophosphatidylcholine (LPC) to produce phosphatidylcholine (PC), the main phospholipid in cellular membranes. In this study, three LPCAT genes from sunflower were identified and the corresponding proteins characterized. These HaLPCAT genes encoded functionally active enzymes that were able to complement a deficient yeast mutant. Moreover, enzymatic assays were carried out using microsomal preparations of the yeast cells. When acyl specificities were measured in the forward reaction, these enzymes exhibited a substrate preference for unsaturated acyl-CoAs, especially for linolenoyl-CoA, while in the reverse reaction, linoleoyl or linolenoyl acyl groups were transferred from PC to acyl-CoA to a similar extent. Expression levels of LPCAT genes were studied revealing distinct tissue-specific expression patterns. In summary, this study suggests that the combined forward and reverse reactions catalyzed by sunflower LPCATs facilitate acyl-exchange between the sn-2 position of PC and the acyl-CoA pool. Sunflower LPCATs displayed different characteristics, which could point to different functionalities, favoring the enrichment of seed triacylglycerols (TAGs) with polyunsaturated fatty acid (PUFA)España, AEI and FEDER Project AGL2017- 83449-R. R

Acyl-CoA:diacylglycerol acyltransferase: Properties, physiological roles, metabolic engineering and intentional control

Acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the last reaction in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG). DGAT activity resides mainly in DGAT1 and DGAT2 in eukaryotes and bifunctional wax ester synthase-diacylglycerol acyltransferase (WSD) in bacteria, which are all membrane-bound proteins but exhibit no sequence homology to each other. Recent studies also identified other DGAT enzymes such as the soluble DGAT3 and diacylglycerol acetyltransferase (EaDAcT), as well as enzymes with DGAT activities including defective in cuticular ridges (DCR) and steryl and phytyl ester synthases (PESs). This review comprehensively discusses research advances on DGATs in prokaryotes and eukaryotes with a focus on their biochemical properties, physiological roles, and biotechnological and therapeutic applications. The review begins with a discussion of DGAT assay methods, followed by a systematic discussion of TAG biosynthesis and the properties and physiological role of DGATs. Thereafter, the review discusses the three-dimensional structure and insights into mechanism of action of human DGAT1, and the modeled DGAT1 from Brassica napus. The review then examines metabolic engineering strategies involving manipulation of DGAT, followed by a discussion of its therapeutic applications. DGAT in relation to improvement of traits of farmed animals is also discussed along with DGATs in various other eukaryotic organisms

Gene coexpression clusters and putative regulatory elements underlying seed storage reserve accumulation in Arabidopsis

Abstract Background In Arabidopsis, a large number of genes involved in the accumulation of seed storage reserves during seed development have been characterized, but the relationship of gene expression and regulation underlying this physiological process remains poorly understood. A more holistic view of this molecular interplay will help in the further study of the regulatory mechanisms controlling seed storage compound accumulation. Results We identified gene coexpression networks in the transcriptome of developing Arabidopsis (Arabidopsis thaliana) seeds from the globular to mature embryo stages by analyzing publicly accessible microarray datasets. Genes encoding the known enzymes in the fatty acid biosynthesis pathway were found in one coexpression subnetwork (or cluster), while genes encoding oleosins and seed storage proteins were identified in another subnetwork with a distinct expression profile. In the triacylglycerol assembly pathway, only the genes encoding diacylglycerol acyltransferase 1 (DGAT1) and a putative cytosolic "type 3" DGAT exhibited a similar expression pattern with genes encoding oleosins. We also detected a large number of putative cis-acting regulatory elements in the promoter regions of these genes, and promoter motifs for LEC1 (LEAFY COTYLEDON 1), DOF (DNA-binding-with-One-Finger), GATA, and MYB transcription factors (TF), as well as SORLIP5 (Sequences Over-Represented in Light-Induced Promoters 5), are overrepresented in the promoter regions of fatty acid biosynthetic genes. The conserved CCAAT motifs for B3-domain TFs and binding sites for bZIP (basic-leucine zipper) TFs are enriched in the promoters of genes encoding oleosins and seed storage proteins. Conclusions Genes involved in the accumulation of seed storage reserves are expressed in distinct patterns and regulated by different TFs. The gene coexpression clusters and putative regulatory elements presented here provide a useful resource for further experimental characterization of protein interactions and regulatory networks in this process.</p

Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions

Background: Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine. Results: Plant LPCATs were expressed in yeast and biochemically characterized. Conclusion: LPCATs can edit acyl composition of phosphatidylcholine through their combined forward and reverse reactions. Significance: Plant LPCATs play a role in editing both sn-positions of PC and remove ricinoleic acid with high selectivity from this lipid

Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content

Top–down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content

Carbon partitioning between oil and carbohydrates in developing oat (Avena sativa L.) seeds

Cereals accumulate starch in the endosperm as their major energy reserve in the grain. In most cereals the embryo, scutellum, and aleurone layer are high in oil, but these tissues constitute a very small part of the total seed weight. However, in oat (Avena sativa L.) most of the oil in kernels is deposited in the same endosperm cells that accumulate starch. Thus oat endosperm is a desirable model system to study the metabolic switches responsible for carbon partitioning between oil and starch synthesis. A prerequisite for such investigations is the development of an experimental system for oat that allows for metabolic flux analysis using stable and radioactive isotope labelling. An in vitro liquid culture system, developed for detached oat panicles and optimized to mimic kernel composition during different developmental stages in planta, is presented here. This system was subsequently used in analyses of carbon partitioning between lipids and carbohydrates by the administration of 14C-labelled sucrose to two cultivars having different amounts of kernel oil. The data presented in this study clearly show that a higher amount of oil in the high-oil cultivar compared with the medium-oil cultivar was due to a higher proportion of carbon partitioning into oil during seed filling, predominantly at the earlier stages of kernel development