Project description:Triacylglycerol (TAG) is the main storage lipid in plant seeds and the major form of plant oil used for food and, increasingly, for industrial and biofuel applications. Several transcription factors, including FUSCA3 (At3g26790, FUS3), are associated with regulation of embryo 3maturation and oil biosynthesis in seeds. However, the ability of FUS3 to increase TAG biosynthesis in other tissues has not been quantitatively examined. Here, we evaluated the ability of FUS3 to activate TAG accumulation in non-seed tissues. Overexpression of FUS3 driven by an estradiol-inducible promoter increased oil contents in Arabidopsis seedlings up to 6% of dry weight; more than 50 fold over controls. Eicosenoic acid, a characteristic fatty acid of Arabidopsis seed oil, accumulated to over 20% of fatty acids in cotyledons and leaves. These large increases depended on added sucrose, although without sucrose TAG increased 3-4 fold. Inducing the expression of FUS3 in tobacco BY2 cells also increased TAG accumulation, and co-expression of FUS3 and diacylglycerol acyltransferase 1 (DGAT1) further increased TAG levels to 4% of dry weight. BY2 cell growth was not altered by FUS3 expression, although Arabidopsis seedling development was impaired, consistent with the ability of FUS3 to induce embryo characteristics in non-seed tissues. Microarrays of Arabidopsis seedlings revealed that FUS3 overexpression increased expression of a higher proportion of genes involved in TAG biosynthesis than genes involved in fatty acid biosynthesis or other lipid pathways. Together these results provide additional insights into FUS3 functions in TAG metabolism and suggest complementary strategies for engineering vegetative oil accumulation.
Project description:FUSCA3 (FUS3) is a B3 domain transcription factor that is a member of the LEAFY COTYLEDON (LEC) group of genes. The LEC genes encode proteins that also include LEC2, a B3 domain factor related to FUS3, and LEC1, a CCAAT box binding factor. LEC1, LEC2 and FUS3 are essential for plant embryo development. We report ChIP-chip experiments using the Affymetrix tiling array to globally map binding sites for FUS3. Fangfang Wang and Sharyn E. Perry (2013) Plant Physiology preview FUSCA3 was expressed by the native promoter and included a C-terminal 10x-c-myc tag. This transgene was able to rescue the fus3-3 mutant. A second transgene (35S:AGL15) was present to allow establishment of stable somatic embryo cultures. ChIP was performed using anti-c-myc antibody (Myc-Tag (9B11) Mouse mAb; Cell Signaling 2276S) on tissue expressing FUS3-myc (three biological replicates) and on three controls of tissue expressing FUS3 (no epitope tag).
Project description:FUSCA3 (FUS3) is a B3 domain transcription factor that is a member of the LEAFY COTYLEDON (LEC) group of genes. The LEC genes encode proteins that also include LEC2, a B3 domain factor related to FUS3, and LEC1, a CCAAT box binding factor. LEC1, LEC2 and FUS3 are essential for plant embryo development. We report ChIP-chip experiments using the Affymetrix tiling array to globally map binding sites for FUS3. Fangfang Wang and Sharyn E. Perry (2013) Plant Physiology preview
Project description:Understanding the mechanisms of transcriptional regulation for genes involved with FA biosynthesis and TAG accumulation in seeds can represent an important step in the development of crops with increased oil content. Among well studied transcription factors (TFs), WRINKLED1 (WRI1), a member of the APETALA2 (AP2) family of TFs, has been characterized as a central regulator of FA biosynthesis in seeds of many plant species. Despite its importance for the process, the detailed mechanisms of WRI1 function in regulating FA biosynthesis in seeds is still not well understood. This study aimed to characterize the gene networks controled by WRI1 during soybean embryo development and its function as central regulator of FA biosynthesis and TAG accumulation in seeds. In this part of the study, we identified genes misregulated in the embryos of the Arabidopsis mutant wrinkled1-1, when compared to wild-type.
Project description:The FUSCA3 (FUS3) transcription factor is considered to be a master regulator of seed maturation because a wide range of seed maturation events are impaired in its defective mutant. To comprehensively identify genes under the control of FUS3, two types of microarray experiments were performed. First, transgenic plants in which FUS3 expression could be induced by the application of estrogen (ESTR) were used to identify the genes up-regulated in young seedlings of Arabidopsis in response to ectopic expression of FUS3. Second, transcriptomes were compared between fus3 mutant and wild type developing seeds. Combining the results of these experiments identified genes under relatively immediate and robust control of FUS3 during seed development. The analyses expanded the range of types of genes under the control of FUS3. The genes positively controlled by FUS3 are not confined to previously known seed maturation-related genes and include those for production of secondary metabolites such as glucosinolates, phenylpropanoids and falvonoids, and primary metabolism such as photosynthesis and fatty acid biosynthesis. Furthermore, several different patterns were identified in the manner of ectopic activation by FUS3 including the kinetics and abscisic acid (ABA) requirement of downstream genes depending on their natures of developmental regulation, suggesting mechanistic diversity of gene regulation by FUS3.
Project description:Desiccation tolerance (DT) allowed seed plants to conquer ecosystems with long periods of limited water availability. This adaptive features allows seeds to remain dried for very long times without losing their ability to germinate. There is little information about all the signaling components required to achieve DT and on how transcription factors (TFs) modulate global DT processes. We performed RNA-seq experiment and carbohydrates profiles of lec1, lec2, fus3 and abi3, as well as their corresponding wild types, at three stages of seed development 15, 17 and 21 DAF (day after open flower) belonging to the seed desiccation period. A complex experimental design approach and regulatory networks prediction were used to identify differentially expressed genes specifically involved in DT process. In order to identify mechanisms involved in the acquisition of DT during seed development, we designed a comparative transcriptomic analysis between the seed desiccation intolerant (DI) mutants lec1-1, abi3-5 and fus3-3, the desiccation tolerant mutant lec2-1 and the desiccation tolerant weak allele of abi3 (abi3-1) with their respective wild type controls. This analysis should allow to identify genes that are differentially expressed in the desiccation intolerant mutants respect to tolerant mutants and WT controls.
Project description:Desiccation tolerance (DT) allowed seed plants to conquer ecosystems with long periods of limited water availability. This adaptive features allows seeds to remain dried for very long times without losing their ability to germinate. There is little information about all the signaling components required to achieve DT and on how transcription factors (TFs) modulate global DT processes. We performed RNA-seq experiment and carbohydrates profiles of lec1, lec2, fus3 and abi3, as well as their corresponding wild types, at three stages of seed development 15, 17 and 21 DAF (day after open flower) belonging to the seed desiccation period. A complex experimental design approach and regulatory networks prediction were used to identify differentially expressed genes specifically involved in DT process.