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In developing soybean seeds, carbon is partitioned between oil, protein and carbohydrates. Here, we demonstrate that suppression of lipase-mediated turnover of triacylglycerols (TAG) during late seed development increases fatty acid content and decreases the presence of undigestible oligosaccharides. During late stages of embryo development, the fatty acid content of soybean seed decreases while the levels of the oligosaccharides raffinose and stachyose increase. Three soybean genes orthologous to the Arabidopsis lipase gene SUGAR-DEPENDENT1 (SDP1) are upregulated at this time. Suppression of these genes resulted in higher oil levels, with lipid levels in the best lines exceeding 24% of seed weight. In addition, lipase-suppressed lines produced larger seeds compared to wild-type plants, resulting in increases of over 20% in total lipid per seed. Levels of raffinose and stachyose were lower in the transgenic lines, with average reductions of 15% in total raffinose family oligosaccharides observed. Despite the increase in oil, protein content was not negatively impacted and trended higher in the transgenic lines. These results are consistent with a role for SDP1 in turning over TAG to supply carbon for other needs, including the synthesis of oligosaccharides, and offer new strategies to further improve the composition of soybean seeds.

Abstract Background Phospholipids are important structural and signaling molecules in plant membranes. Some fluorescent dyes can stain general lipids of membranes, but labeling and visualization of specific lipid classes have yet to be developed for most components of the membrane. New techniques for visualizing membrane lipids are needed to further delineate their dynamic structural and signaling roles in plant cells. In this study we examined whether propargylcholine, a bioortholog of choline, can be used to label the major membrane lipid, phosphatidylcholine, and other choline phospholipids in plants. We established that propargylcholine is readily taken up by roots, and that its incorporation is not detrimental to plant growth. After plant tissue is harvested and fixed, a click-chemistry reaction covalently links the alkyne group of propargylcholine to a fluorescently-tagged azide, resulting in specific labeling of choline phospholipids. Results Uptake of propargylcholine, followed by click chemistry with fluorescein or Alexa Fluor 594 azide was used to visualize choline phospholipids in cells of root, leaf, stem, silique and seed tissues from Arabidopsis thaliana. Co-localization with various subcellular markers indicated coinciding fluorescent signals in cell membranes, such as the tonoplast and the ER. Among different cell types in the leaf epidermis, guard cells displayed strong labeling. Mass spectrometry-based lipidomic analysis of the various plant tissues revealed that incorporation of propargylcholine was strongest in roots with approximately 50% of total choline phospholipids being labeled, but it was also incorporated in the other tissues including seeds. Phospholipid profiling confirmed that, in each tissue analyzed, incorporation of the bioortholog had little impact on the pool of choline plus choline-like phospholipids or other lipid species. Conclusion We developed and validated a click-chemistry based method for fluorescence imaging of choline phospholipids using a bioortholog of choline, propargylcholine, in various cell-types and tissues from Arabidopsis. This click-chemistry method provides a direct way to metabolically tag and visualize specific lipid molecules in plant cells. This work paves the way for future studies addressing in situ localization of specific lipids in plants.

Bacterial glycan structures on cell surfaces are critical for cellcell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3-Cmethyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependentSAM: C-methyltransferase, and NADPH-dependent CDP-3-C-methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3-C-methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3-C-methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3-C-methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C-methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2-1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3-C-methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus. [ABSTRACT FROM AUTHOR]