We investigated the role of the oilbody proteins in developing and germinating Arabidopsis thaliana seeds. Seed oilbodies are simple organelles comprising a matrix of triacylglycerol surrounded by a phospholipid monolayer embedded and covered with unique proteins called oleosins. Indirect observations have suggested that oleosins maintain oilbodies as small single units preventing their coalescence during seed desiccation. To understand the role of oleosins during seed development or germination, we created lines of Arabidopsis in which a major oleosin is ablated or severely attenuated. This was achieved using RNA interference techniques and through the use of a T-DNA insertional event, which appears to interrupt the major (18 kD) seed oleosin gene of Arabidopsis and results in ablation of expression. Oleosin suppression resulted in an aberrant phenotype of embryo cells that contain unusually large oilbodies that are not normally observed in seeds. Changes in the size of oilbodies caused disruption of storage organelles, altering accumulation of lipids and proteins and causing delay in germination. The aberrant phenotypes were reversed by reintroducing a recombinant oleosin. Based on this direct evidence, we have shown that oleosins are important proteins in seed tissue for controlling oilbody structure and lipid accumulation.

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Background: Triacylglycerol (TAG) can be formed via an acyl-CoA-dependent or acyl-CoA-independent pathway. Results: Overexpressing particular flax phospholipid:diacylglycerol acyltransferase (PDAT) genes in yeast and Arabidopsis resulted in an enhanced proportion of ␣-linolenic acid (ALA) in TAG. Conclusion: Certain PDATs have the unique ability to efficiently channel ALA into TAG. Significance: The identified PDATs will benefit future projects aimed at producing oils with enhanced polyunsaturated fatty acid content.

Acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20) is a membrane protein present mainly in the endoplasmic reticulum. It catalyzes the final and committed step in the biosynthesis of triacylglycerol, which is the principal repository of fatty acids for energy utilization and membrane formation. Two distinct family members of acyl-CoA:diacylglycerol acyltransferase, known as DGAT1 and DGAT2, have been characterized in different organisms, including mammals, fungi, and plants. In this study, we characterized the functional role and topological orientation of signature motifs in yeast (Saccharomyces cerevisiae) DGAT2 using mutagenesis in conjunction with chemical modification. Our data provide evidence that both the N and C termini are oriented toward the cytosol and have different catalytic roles. A highly conserved motif, 129YFP131, and a hydrophilic segment exclusive to yeast DGAT2 reside in a long endoplasmic reticulum luminal loop following the first transmembrane domain and play an essential role in enzyme catalysis. In addition, the strongly conserved His195 within the motif HPHG, which may play a role in the active site of DGAT2, is likely embedded in the membrane. These results indicate some similarities to the topology model of murine DGAT2 but also reveal striking differences suggesting that the topological organization of DGAT2 is not ubiquitously conserved.

Insulin and leptin have overlapping effects in the control of energy homeostasis, but the molecular basis of this synergism is unknown. Insulin signals through a receptor tyrosine kinase that phosphorylates and activates the docking proteins IRSs (insulin receptor substrates), whereas the leptin receptor and its associated protein tyrosine kinase JAK2 (Janus kinase 2) mediate phosphorylation and activation of the transcription factor STAT3 (signal transducer and activator of transcription). Here, we present evidence for the integration of leptin and insulin signals in the hypothalamus. Insulin induced JAK2 tyrosine phosphorylation, leptin receptor phosphorylation which, in the presence of leptin, augmented the interaction between STAT3 and this receptor. Insulin also increased the leptin-induced phosphorylation of STAT3 and its activation. These results indicate that insulin modulates the leptin signal transduction pathway, and may provide a molecular basis for the coordinated effects of insulin and leptin in feeding behavior and weight control. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.