T9C 1T4 (A.E.-M., S.S.)Brassica napus is an allotetraploid (AACC) formed from the fusion of two diploid progenitors, Brassica rapa (AA) and Brassica oleracea (CC). Polyploidy and genome-wide rearrangement during the evolution process have resulted in genes that are present as multiple homologs in the B. napus genome. In this study, three B. napus homologous genes encoding endoplasmic reticulumbound sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) were identified and characterized. Although the three GPAT4 homologs share a high sequence similarity, they exhibit different expression patterns and altered epigenetic features. Heterologous expression in yeast further revealed that the three BnGPAT4 homologs encoded functional GPAT enzymes but with different levels of polypeptide accumulation. Complementation of the Arabidopsis (Arabidopsis thaliana) gpat4 gpat8 double mutant line with individual BnGPAT4 homologs suggested their physiological roles in cuticle formation. Analysis of gpat4 RNA interference lines of B. napus revealed that the BnGPAT4 deficiency resulted in reduced cutin content and altered stomatal structures in leaves. Our results revealed that the BnGPAT4 homologs have evolved into functionally divergent forms and play important roles in cutin synthesis and stomatal development.Polyploidy has long been considered a prominent evolutionary force for flowering plants ( Udall and Wendel, 2006). Over 95% of the lineages of angiosperms have undergone at least one event of polyploidization over their evolutionary time, suggesting that most of the existing flowering plants evolved from ancient polyploids (Bennett and Leitch, 1997;Chen, 2007;Gaeta et al., 2007). Polyploidy, along with genomic segmental duplications, could benefit plants by increasing overall gene expression levels and cell sizes and providing sources for novel variants and genome "buffering" of deleterious mutations (Udall and Wendel, 2006). Genes duplicated by such events could undergo three primary evolutionary fates over the long term (Wendel, 2000;Blanc and Wolfe, 2004;Lukens et al., 2004;Whittle and Krochko, 2009): (1) pseudogenization (loss or silencing), whereby duplicated genes with redundant functions accumulate deleterious mutations and are eventually lost without detrimental effects on plant fitness; (2) neofunctionalization, whereby some redundant genes develop new adaptive functions by positive Darwinian selection; and (3) subfunctionalization, a process in which the ancestral gene functions become subdivided among the duplicated genes.Brassica napus (AACC; n = 19) is an allotetraploid oilseed crop that evolved from the hybridization of two diploid progenitors, Brassica rapa (AA; n = 10) and Brassica oleracea (CC; n = 9) during human cultivation (over 10,000 years ago; U, 1935;Cheung et al., 2009). The Brassica species are closely related to the model plant Arabidopsis (Arabidopsis thaliana), all of which belong to the same tribe (Brassiceae) and share a common recent ancestry (20 million years ago; Yang et al., 1999). Comparative...

The canola industry generates more than $11 billion of yearly income to the Canadian economy. One problem of meal quality is the dark polyphenolic pigments that accumulate in the seed coat. Seed coat-specific promoters are a pre-requisite to regulate the genes involved in seed coat development and metabolism. The beta-glucuronidase (GUS) reporter gene was used to test an Arabidopsis promoter in developing and mature seeds of canola (Brassica napus). The promoter tested is the regulatory region of the laccase gene (AtLAC15) from Arabidopsis thaliana. The AtLAC15 promoter::GUS construct was inserted into canola double haploid line DH12075 using Agrobacterium-mediated transformation. Southern blot analysis using a 536 bp GUS probe showed variation among the transformed plants in the T-DNA copy numbers and the position of the insertion in their genomes. Histochemical assay of the GUS enzyme in different tissues (roots, leaves, stem, pollen grains, flowers, siliques, embryos and seed coats) showed ascending GUS activity only in the seed coat from 10 days after pollination (DAP) to the fully mature stage (35 DAP). GUS stain was observed in the mucilage cell layer, in the outer integument layer of the seed coat but not in the inner integument. The AtLAC15 promoter exhibited a specificity and expression level that is useful as a seed coat-specific promoter for canola.

In search for seed coat-specific promoters for canola (Brassica napus), transgenic plants carrying a 2,121 bp fragment of Arabidopsis thaliana At4g12960 promoter (AtGILTpro) fused to the uidA reporter gene (GUS) were generated. Out of 7 independent events in transgenic canola plants raised, 2 exhibited GUS activity exclusively in the outer integument of the seed coat. GUS activity in other tissues was also observed in the remaining five transformants. Therefore, the AtGILT promoter can be used as a canola seed coat outer integument-specific promoter after the generation and selection of desired transformants from several transgenic lines.

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