Canola (Brassica napus) oil has immense economic value due to its use as food. The production of biodiesel has led to the recent increase in the demand for canola oil, and further reinforced the need to improve the oil content and composition of canola seeds. The content and quality of canola oil is dependent on the process of fatty acid biosynthesis and accumulation of triacylglycerols (TAGs) during seed development. The biochemical pathways leading to the synthesis of fatty acids and TAGs are thoroughly understood, and many of the genes encoding the metabolic enzymes involved in this pathway have been isolated and functionally characterized. Despite comprehensive understanding of fatty acid and TAG biosynthesis, alternate factors and genes involved in the modification of seed oil content and composition are being investigated. This has initiated a new sector of research focusing on a series of transcription factors: LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON2 (LEC2), FUSCA3 (FUS3) and WRINKLED1 (WRI1), which play a key role during embryo and seed development. Previous studies in Arabidopsis have demonstrated that the noted transcription factors influence the synthesis and accumulation of oil during seed development. These findings have prompted the design of two studies with the objective of further characterizing the role of these genes. In the first study, a homolog of Arabidopsis LEC1 was isolated from Brassica napus (Bn) microspore derived embryos. BnLEC1 contained the same distinct features identified in LEC1 genes from other plant species. BnLEC1 was inserted into an expression vector, which was subsequently used to transform Arabidopsis plants. The transgenic lines were characterized by differences in seed oil composition, and one line showed a reduction in the number of seeds counted per silique. Expression analysis revealed that the transgene was not expressed in the transgenic lines. To account for the discrepant findings, the possibility of endogenous gene silencing mechanisms suppressing transgene expression has been discussed. As an extension of this research, a second study assessed the differential expression of LEC1, LEC2, FUS3 and WRI1 in double haploid Brassica napus lines characterized by different levels of seed oil content. The expression level of the noted genes was determined at 7, 14, 21 and 28 days after pollination. Numerous changes in the transcript level were observed, but the trends were not consistent among high and low oil content lines.

Canola (Brassica napus) oil has immense economic value due to its use as food. The production of biodiesel has led to the recent increase in the demand for canola oil, and further reinforced the need to improve the oil content and composition of canola seeds. The content and quality of canola oil is dependent on the process of fatty acid biosynthesis and accumulation of triacylglycerols (TAGs) during seed development. The biochemical pathways leading to the synthesis of fatty acids and TAGs are thoroughly understood, and many of the genes encoding the metabolic enzymes involved in this pathway have been isolated and functionally characterized. Despite comprehensive understanding of fatty acid and TAG biosynthesis, alternate factors and genes involved in the modification of seed oil content and composition are being investigated. This has initiated a new sector of research focusing on a series of transcription factors: LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON2 (LEC2), FUSCA3 (FUS3) and WRINKLED1 (WRI1), which play a key role during embryo and seed development. Previous studies in Arabidopsis have demonstrated that the noted transcription factors influence the synthesis and accumulation of oil during seed development. These findings have prompted the design of two studies with the objective of further characterizing the role of these genes. In the first study, a homolog of Arabidopsis LEC1 was isolated from Brassica napus (Bn) microspore derived embryos. BnLEC1 contained the same distinct features identified in LEC1 genes from other plant species. BnLEC1 was inserted into an expression vector, which was subsequently used to transform Arabidopsis plants. The transgenic lines were characterized by differences in seed oil composition, and one line showed a reduction in the number of seeds counted per silique. Expression analysis revealed that the transgene was not expressed in the transgenic lines. To account for the discrepant findings, the possibility of endogenous gene silencing mechanisms suppressing transgene expression has been discussed. As an extension of this research, a second study assessed the differential expression of LEC1, LEC2, FUS3 and WRI1 in double haploid Brassica napus lines characterized by different levels of seed oil content. The expression level of the noted genes was determined at 7, 14, 21 and 28 days after pollination. Numerous changes in the transcript level were observed, but the trends were not consistent among high and low oil content lines.

Douglas-fir (Pseudotsuga menziesii) is an economically important softwood that is clonally propagated for reforestation purposes by somatic embryogenesis. The molecular basis of embryogenesis in conifers is largely unknown and this prevents progress in somatic embryogenesis protocols. In angiosperms, the LEAFY COTYLEDON1 (LEC1) gene, encoding the HAP3 subunit of the eukaryotic CCAAT box-binding factor, is important in embryo formation, and necessary for somatic embryogenesis. A candidate gene strategy was employed to isolate the Douglas-fir LEC1 homologue, PmLEC1, via the polymerase chain reaction (PCR) with degenerate primers based on the Arabidopsis conserved domain, and the full-length cDNA sequence was obtained by rapid amplification of cDNA ends-PCR (RACE-PCR). The putative protein sequence shared high sequence identity with Arabidopsis LEC1. Northern analysis and quantitative real-time PCR indicate that this is an embryo-specific gene, expressed with the highest abundance during early embryogenesis. Antibodies were raised against a synthetic 18-amino acid PmLEC1 peptide, and in contrast to mRNA expression, Western blotting shows that PmLEC1 protein expression persists until the seedling stage. To gain insight into modulation of PmLEC1 expression and its inducibility in mature tissues, stress and hormone treatments were performed on mature seed and the promoter sequence was isolated by genome walking. Sorbitol, mannitol and 2,4-epibrassinolide were found to significantly up-regulate PmLEC1 expression. The PmLEC1 promoter contains a 5’ UTR intron with numerous enhancer elements, and factors that bind to these elements mediate responses to auxin, UV light and developmental cues, osmotic stress, biotic stress, and tissue culture. Some of the regulatory elements are binding sites for seed-specific transcription factors that are well known from angiosperms, providing new evidence that AGL15, ABI3 and VP1 proteins have a direct role on LEC1 expression. In investigating the embryogenic capacity of PmLEC1, ectopic expression of PmLEC1 in the embryo lethal Arabidopsis lec1-1 null mutant complemented the mutation and permitted the production of viable, desiccation tolerant seeds. In addition, transgenic seedlings produced embryo-like structures from vegetative organs and expressed seed-specific genes. In wild type plants, ectopic expression of PmLEC1 resulted in a bushy phenotype but expression of seed-specific genes was not observed. Taken together, these results show that PmLEC1 is an embryo-specific gene with an essential role throughout embryogenesis, and PmLEC1 expression may be induced in mature seeds by stress and hormone treatments. Because mature seeds show only trace amounts of PmLEC1 transcripts and Douglas-fir somatic embryogenesis can only be induced from immature embryos, this information provides useful insight into initiation of embryogenesis from vegetative tissues. The identification of binding sites for transcription factors known from angiosperms in the promoter region of PmLEC1 has revealed the identity of several genes which are expected to play pivotal roles in conifer embryogenesis.