Overexpression of phospholipid: diacylglycerol acyltransferase in Brassica napus results in changes in lipid metabolism and oil accumulation.

The regulation of lipid metabolism in oil seeds is still not fully understood and increasing our knowledge in this regard is of great economic, as well as intellectual, importance. Oilseed rape (Brassica napus) is a major global oil crop where increases in triacylglycerol (TAG) accumulation have been achieved by overexpression of relevant biosynthetic enzymes. In this study, we expressed Arabidopsis phospholipid: diacylglycerol acyltransferase (PDAT1), one of the two major TAG-forming plant enzymes in B. napus DH12075 to evaluate its effect on lipid metabolism in developing seeds and to estimate its flux control coefficient. Despite several-fold increase in PDAT activity, seeds of three independently generated PDAT transgenic events showed a small but consistent decrease in seed oil content and had altered fatty acid composition of phosphoglycerides and TAG, towards less unsaturation. Mass spectrometry imaging of seed sections confirmed the shift in lipid compositions and indicated that PDAT overexpression altered the distinct heterogeneous distributions of phosphatidylcholine (PC) molecular species. Similar, but less pronounced, changes in TAG molecular species distributions were observed. Our data indicate that PDAT exerts a small, negative, flux control on TAG biosynthesis and could have under-appreciated effects in fine-tuning of B. napus seed lipid composition in a tissue-specific manner. This has important implications for efforts to increase oil accumulation in similar crops.

Comparative Transcriptomics Analysis of Brassica napus L. during Seed Maturation Reveals Dynamic Changes in Gene Expression between Embryos and Seed Coats and Distinct Expression Profiles of Acyl-CoA-Binding Proteins for Lipid Accumulation.

Production of vegetable oils is a vital agricultural resource and oilseed rape (Brassica napus) is the third most important oil crop globally. Although the regulation of lipid biosynthesis in oilseeds is still not fully defined, the acyl-CoA-binding proteins (ACBPs) have been reported to be involved in such metabolism, including oil accumulation, in several plant species. In this study, progressive changes in gene expression in embryos and seed coats at different stages of seed development were comprehensively investigated by transcriptomic analyses in B. napus, revealing dynamic changes in the expression of genes involved in lipid biosynthesis. We show that genes encoding BnACBP proteins show distinct changes in expression at different developmental stages of seed development and show markedly different expression between embryos and seed coats. Both isoforms of the ankyrin-repeat BnACBP2 increased during the oil accumulation period of embryo development. By contrast, the expression of the three most abundant isoforms of the small molecular mass BnACBP6 in embryos showed progressive reduction, despite having the highest overall expression level. In seed coats, BnACBP3, BnACBP4 and BnACBP5 expression remained constant during development, whereas the two major isoforms of BnACBP6 increased, contrasting with the data from embryos. We conclude that genes related to fatty acid and triacylglycerol biosynthesis showing dynamic expression changes may regulate the lipid distribution in embryos and seed coats of B. napus and that BnACBP2 and BnACBP6 are potentially important for oil accumulation.

Increase in lysophosphatidate acyltransferase activity in oilseed rape (Brassica napus) increases seed triacylglycerol content despite its low intrinsic flux control coefficient.

Lysophosphatidate acyltransferase (LPAAT) catalyses the second step of the Kennedy pathway for triacylglycerol (TAG) synthesis. In this study we expressed Trapaeolum majus LPAAT in Brassica napus (B. napus) cv 12075 to evaluate the effects on lipid synthesis and estimate the flux control coefficient for LPAAT. We estimated the flux control coefficient of LPAAT in a whole plant context by deriving a relationship between it and overall lipid accumulation, given that this process is a exponential. Increasing LPAAT activity resulted in greater TAG accumulation in seeds of between 25% and 29%; altered fatty acid distributions in seed lipids (particularly those of the Kennedy pathway); and a redistribution of label from 14 C-glycerol between phosphoglycerides. Greater LPAAT activity in seeds led to an increase in TAG content despite its low intrinsic flux control coefficient on account of the exponential nature of lipid accumulation that amplifies the effect of the small flux increment achieved by increasing its activity. We have also developed a novel application of metabolic control analysis likely to have broad application as it determines the in planta flux control that a single component has upon accumulation of storage products.

Using lipidomics to reveal details of lipid accumulation in developing seeds from oilseed rape (Brassica napus L.).

With dwindling available agricultural land, concurrent with increased demand for oil, there is much current interest in raising oil crop productivity. We have been addressing this issue by studying the regulation of oil accumulation in oilseed rape (Brassica napus L). As part of this research we have carried out a detailed lipidomic analysis of developing seeds. The molecular species distribution in individual lipid classes revealed quite distinct patterns and showed where metabolic connections were important. As the seeds developed, the molecular species distributions changed, especially in the period of early (20days after flowering, DAF) to mid phase (27DAF) of oil accumulation. The patterns of molecular species of diacylglycerol, phosphatidylcholine and acyl-CoAs were used to predict the possible relative contributions of diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase to triacylglycerol production. Our calculations suggest that DGAT may hold a more important role in influencing the molecular composition of TAG. Enzyme selectivity had an important influence on the final molecular species patterns. Our data contribute significantly to our understanding of lipid accumulation in the world's third most important oil crop.

Spatial and Temporal Mapping of Key Lipid Species in Brassica napus Seeds.

The regulation of lipid synthesis in oil seeds is still not fully understood. Oilseed rape (Brassica napus) is the third most productive vegetable oil crop on the global market; therefore, increasing our understanding of lipid accumulation in oilseed rape seeds is of great economic, as well as intellectual, importance. Matrix-assisted laser/desorption ionization-mass spectrometry imaging (MALDI-MSI) is a technique that allows the mapping of metabolites directly onto intact biological tissues, giving a spatial context to metabolism. We have used MALDI-MSI to study the spatial distribution of two major lipid species, triacylglycerols and phosphatidylcholines. A dramatic, heterogenous landscape of molecular species was revealed, demonstrating significantly different lipid compositions between the various tissue types within the seed. The embryonic axis was found to be particularly enriched in palmitic acid, while the seed coat/aleurone layer accumulated vaccenic, linoleic, and α-linoleic acids. Furthermore, the lipid composition of the inner and outer cotyledons differed from each other, a remarkable discovery given the supposed identical functionality of these two tissues. Triacylglycerol and phosphatidylcholine molecular species distribution was analyzed through a developmental time series covering early seed lipid accumulation to seed maturity. The spatial patterning of lipid molecular species did not vary significantly during seed development. Data gathered using MALDI-MSI was verified through gas chromatography analysis of dissected seeds. The distinct lipid distribution profiles observed imply differential regulation of lipid metabolism between the different tissue types of the seed. Further understanding of this differential regulation will enhance efforts to improve oilseed rape productivity and quality.

Acyl-Trafficking During Plant Oil Accumulation.

Vegetable oils are an extremely important agricultural commodity. Their use has risen inexorably for the last 50 years and will undoubtedly be even more prevalent in the future. They have a role not only in foodstuffs but also as renewable chemicals. However, our understanding of their metabolism, and particularly its control, is incomplete. In this article we highlight current knowledge and its deficiencies. In particular, we focus on the important role that phosphatidylcholine plays in lipid accumulation and in influencing the quality of the vegetable oils produced.