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Acetyl-TAG (3-acetyl-1,2-diacylglycerol), unique triacylglycerols (TAG) possessing an acetate group at the sn-3 position, exhibit valuable properties, such as reduced viscosity and freezing points. Previous attempts to engineer acetyl-TAG production in oilseed crops did not achieve the high levels found in naturally producing Euonymus seeds. Here, we demonstrate the successful generation of camelina and pennycress transgenic lines accumulating nearly pure acetyl-TAG at 93 mol% and 98 mol%, respectively. These ultrahigh acetyl-TAG synthesizing lines were created using gene-edited FATTY ACID ELONGASE1 (FAE1) mutant lines as an improved genetic background to increase levels of acetyl-CoA available for acetyl-TAG synthesis mediated by the expression of EfDAcT, a high-activity diacylglycerol acetyltransferase isolated from Euonymus fortunei. Combining EfDAcT expression with suppression of the competing TAG-synthesizing enzyme DGAT1 further enhanced acetyl-TAG accumulation. These ultrahigh levels of acetyl-TAG exceed those in earlier engineered oilseeds and are equivalent or greater than those in Euonymus seeds. Imaging of lipid localization in transgenic seeds revealed that the low amounts of residual TAG were mostly confined to the embryonic axis. Similar spatial distributions of specific TAG and acetyl-TAG molecular species, as well as their probable diacylglycerol (DAG) precursors, provide additional evidence that acetyl-TAG and TAG are both synthesized from the same tissue-specific DAG pools. Remarkably, this ultrahigh production of acetyl-TAG in transgenic seeds exhibited minimal negative effects on seed properties, highlighting the potential for production of designer oils required for economical biofuel industries. [ABSTRACT FROM AUTHOR]

Isotopic labeling studies of primary metabolism frequently utilize GC/MS to quantify (13)C in protein-hydrolyzed amino acids. During processing some amino acids are degraded, which reduces the size of the measurement set. The advent of high-resolution mass spectrometers provides a tool to assess molecular masses of peptides with great precision and accuracy and computationally infer information about labeling in amino acids. Amino acids that are isotopically labeled during metabolism result in labeled peptides that contain spatial and temporal information that is associated with the biosynthetic origin of the protein. The quantification of isotopic labeling in peptides can therefore provide an assessment of amino acid metabolism that is specific to subcellular, cellular, or temporal conditions. A high-resolution orbital trap was used to quantify isotope labeling in peptides that were obtained from unlabeled and isotopically labeled soybean embryos and Escherichia coli cultures. Standard deviations were determined by estimating the multinomial variance associated with each element of the m/z distribution. Using the estimated variance, quantification of the m/z distribution across multiple scans was achieved by a nonlinear fitting approach. Observed m/z distributions of uniformly labeled E. coli peptides indicated no significant differences between observed and simulated m/z distributions. Alternatively, amino acid m/z distributions obtained from GC/MS were convolved to simulate peptide m/z distributions but resulted in distinct profiles due to the production of protein prior to isotopic labeling. The results indicate that peptide mass isotopologue measurements faithfully represent mass distributions, are suitable for quantification of isotope-labeling-based studies, and provide additional information over existing methods.

Summary: Mosses hold a unique position in plant evolution and are crucial for protecting natural, long‐term carbon storage systems such as permafrost and bogs. Due to small stature, mosses grow close to the soil surface and are exposed to high levels of CO2, produced by soil respiration. However, the impact of elevated CO2 (eCO2) levels on mosses remains underexplored.We determined the growth responses of the moss Physcomitrium patens to eCO2 in combination with different nitrogen levels and characterized the underlying physiological and metabolic changes.Three distinct growth characteristics, an early transition to caulonema, the development of longer, highly pigmented rhizoids, and increased biomass, define the phenotypic responses of P. patens to eCO2. Elevated CO2 impacts growth by enhancing the level of a sugar signaling metabolite, T6P. The quantity and form of nitrogen source influences these metabolic and phenotypic changes. Under eCO2, P. patens exhibits a diffused growth pattern in the presence of nitrate, but ammonium supplementation results in dense growth with tall gametophores, demonstrating high phenotypic plasticity under different environments.These results provide a framework for comparing the eCO2 responses of P. patens with other plant groups and provide crucial insights into moss growth that may benefit climate change models. [ABSTRACT FROM AUTHOR]