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The major storage lipids in oil-accumulating seeds such as Arabidopsis thaliana are triacylglycerols (TAGs), esters of glycerol and fatty acids (FAs). During seed development, incoming sucrose is cleaved to hexoses by cell wall-bound invertase (cwINV) and transported into cells, or sucrose transported into the cell is cleaved into UDP-glucose and fructose by sucrose synthase (SUS) in the cytoplasm. De novo synthesis of FAs occurs in the plastid, which imports intermediates of sugar metabolism such as glucose 6-phosphate (Glu6P) and phosphoenolpyruvate (PEP) from the cytoplasm; the metabolic pathway for the synthesis of FAs in plants has been extensively studied (Ohlrogge and Browse, 1995; Rawsthorne, 2002; Nishida, 2005). The C16:0, C16:1 and C18:1 FAs synthesized in plastids (Nishida, 2005) are further elongated and/or desaturated in the endoplasmic reticulum (ER), where the assembly of TAGs occurs through stepwise acylation of a glycerol backbone (Ohlrogge and Browse, 1995). The final step of TAG assembly is catalyzed by acyl-CoA:diacylglycerol acyltransferase (TAG1/DGAT1; Routaboul et al., 1999; Zou et al., 1999), and TAGs are finally coated with oleosins to form oil bodies (Huang, 1996).
In Arabidopsis, developing seeds first accumulate starch, which is later degraded, and oil accumulation occurs with the accumulation of seed storage proteins during the late maturation phase (Baud et al., 2002; Ruuska et al., 2002; Hills, 2004). LEAFY COTYLEDON1 (LEC1) and LEC2, FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE3 (ABI3), function as the master regulators of embryogenesis and seed maturation (Parcy et al., 1997; Lotan et al., 1998; To et al., 2006). Mutants of these LEC-type transcription factors show defective seed maturation with reduced accumulation of seed storage proteins and TAGs, while their ectopic expression in early vegetative seedlings causes the synthesis of seed storage compounds (Kagaya et al., 2005; Santos et al., 2005; Braybrook et al., 2006).
Seeds of the wrinkled1 (wri1) mutant show 80% reduction in TAGs compared with the wild type and accumulate sucrose (Focks and Benning, 1998). WRI1 is an APETALA2 (AP2)-type transcription factor with two AP2 DNA-binding domains (Cernac and Benning, 2004). The ACTIVATOR of SPOmin:LUC1 (ASML1) identified from an enhancer activation-tagged mutant that overexpresses a sugar-inducible luciferase (LUC) reporter gene was identical with WRI1 (Masaki et al., 2005). Expression of WRI1 occurs during seed development (Cernac and Benning, 2004; Masaki et al., 2005), and expression of genes involved in glycolysis and lipid synthesis in developing seeds is compromised in wri1 mutants (Ruuska et al., 2002). WRI1 functions downstream of LEC1 and LEC2, and ectopic expression of LEC1, LEC2, or WRI1 in vegetative tissues up-regulates the expression of genes involved in FA synthesis (Baud et al., 2007; Mu et al., 2008). To understand the role of WRI1 in the regulation of seed oil accumulation and in seed maturation and germination it is important to identify direct targets of WRI1 and its binding site sequences. Although DNA binding of one of the AP2-type transcription factors, AINEGMENTA (ANT; Elliott et al., 1996; Klucher et al., 1996), has been characterized (Nole-Wilson and Krizek, 2000; Krizek, 2003), direct target genes of ANT and other AP2-type transcription factors have not been identified so far.
In the present study we show that many genes involved in various steps of FA synthesis in plastids are the direct targets of WRI1 during seed maturation. The proximal upstream regions of these genes contain a conserved sequence, designated as the AW-box, to which WRI1 binds. Many WRI1 targets involved in FA synthesis are genes with TATA-less promoters, and many AW-boxes are located near the transcription start site (TSS) and in the 5′-untranslated region (5′-UTR) of these genes. The AW-boxes located at TSS and in the 5′-UTR of the Pl-Pkβ1 gene are found to be necessary for activation by WRI1 and expression during seed maturation.
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