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Genes involved in the de novo synthesis of FA in plastids formed the largest group of metabolic genes up-regulated by WRI1 overexpression. Proteins encoded by these genes participate in various steps of the uptake of glycolytic intermediates into plastids, conversion to acetyl-CoA and malonyl-CoA, and the synthesis of FAs from them. In addition, genes involved in the synthesis of lipoic acid, a cofactor of PDH, are also activated in both Enh-WRI1 and 35S-WRI1 plants. Similar results were reported by Baud et al. (2007). Consistent with the expression levels of WRI1 mRNA (Figure 1b), enhanced expression of genes involved in FA synthesis was more prominent in Enh-WRI1 compared with 35S-WRI1 (Figure 2a). On the other hand, mRNA levels of genes involved in FA synthesis were reduced in wri1-10.
Expression of the LUC reporter genes with the proximal upstream regions of Pl-PKβ1, BCCP2, ACP1, and KAS1 in protoplasts was transactivated by co-expression of WRI1 (Figure 3a), and recombinant WRI1 showed specific binding to the unique sequence (AW-box) within the upstream regions (Figure 4). Mutations in two AW-boxes in the upstream region of Pl-PKβ1 abolished the WRI1-binding in vitro, transactivation by WRI1 in protoplasts, and expression during seed development (Figure 6). Altogether, these results indicate that genes involved in FA synthesis in plastids are among the direct targets of WRI1 during seed development. Expression patterns of these genes involved in FA synthesis during seed development are quite similar to that of WRI1 (Figure 3d; Baud et al., 2007; Ruuska et al., 2002).
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