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- Supporting Information
The phytohormone ABA is required to protect embryos during the desiccation stage of seed development by activating downstream genes in concert with the transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3)/VIVIPAROUS 1 (VP1; Finkelstein et al., 2002). Seeds of abi3/vp1 mutants are characterized by reduced amounts of storage proteins, sensitivity to desiccation, and precocious germination (Ooms et al., 1993; Nambara et al., 1994; Parcy et al., 1994, 1997).
The ABI3/VP1 protein has three functional domains, B1, B2 and B3, which are highly conserved among various plant species (Hill et al., 1996; Suzuki et al., 1997), including Physcomitrella patens ABI3 (PpABI3; Marella et al., 2006). The B1 and B2 domains are involved in a complex with the bZIP transcription factor ABI5 (Nakamura et al., 2001) and with the ABA-responsive element (ABRE; Hill et al., 1996), respectively. Additionally, the B2 domain also regulates nuclear localization (Marella & Quatrano, 2007), while the B3 domain binds DNA, specifically the RY/Sph (RY) promoter element (CATGCA; Suzuki et al., 1997; Ezcurra et al., 2000). These three domains of ABI3/VP1 along with specific cis-elements and transcription factors such as bZIPs, regulate the expression of seed-specific genes, for example, the wheat Em gene (Vasil et al., 1995) and the maize C1 gene (Hattori et al., 1992).
In P. patens also, ABA treatment regulates a large set of genes as demonstrated by microarray analysis (Machuka et al., 1999; Cuming et al., 2007; Richardt et al., 2010). Indeed, the ABA-signaling pathway was reported to be present in P. patens, based on the activation of the wheat Em promoter and the PpLEA1 (the P. patens homolog of Em) promoter by exogenous ABA treatment (Knight et al., 1995; Kamisugi & Cuming, 2005) and its activation is performed through an ACGT-core ABRE (Kamisugi & Cuming, 2005; Sakata et al., 2010). PpABI3A was shown to activate synergistically the PpLEA1 promoter in concert with ABA (Marella et al., 2006; Khandelwal et al., 2010) and to partially complement the phenotypes of the Arabidopsis abi3-6 mutant such as ABA insensitivity of germination, the color of seeds, and the expression of seed maturation genes (MAT), but it failed to restore the expression of the LEA genes through the ACGT-core ABRE (Marella et al., 2006). On the other hand, a previous report has shown that the napA (the Brassica napus homolog of MAT genes) promoter is activated not only through an ACGT-core ABRE but also an ACTT-core ABRE (5′-GCCACTTGTC-3′), called distB for the transactivation by ABI3 both with and without ABA (Ezcurra et al., 2000).
LEAFY COTYLEDON1 (LEC1) was also found to play central roles in the regulation of embryo maturation in seeds (Meinke et al., 1994; West et al., 1994), similar to ABI3. Ectopically overexpressing LEC1 can induce embryo development in vegetative cells (Lotan et al., 1998). LEC1 encodes a homolog of yeast HAP3 subunit of the CCAAT box binding factor (CBF)/nuclear factor Y (NF-Y), which is an evolutionarily conserved transcription factor that contains three subunits, HAP2 (CBF-B/NF-YA), HAP3 (CBF-A/NF-YB), and HAP5 (CBF-C/NF-YC; Li et al., 1992). The complex forms through an initial interaction between HAP3 and HAP5, which then allows the formation of a heterotrimer with HAP2. The heterotrimer then binds to a CCAAT element with very high specificity and affinity (Romier et al., 2003).
Each subunit of the NF-Y complex is encoded by gene families in angiosperms (Stephenson et al., 2007; Siefers et al., 2009). For example, the Arabidopsis genome encodes 10 NF-YA, 13 NF-YB, and 13 NF-YC paralogs (Siefers et al., 2009). Several other NF-Y subunits have been implicated in specific developmental processes in angiosperms, including nodule formation (Combier et al., 2006), flowering-time regulation (Ben-Naim et al., 2006; Wenkel et al., 2006; Cai et al., 2007; Kumimoto et al., 2010; Li et al., 2011), ABA/blue light response (Warpeha et al., 2007; Yamamoto et al., 2009), stress response (Chen et al., 2007; Nelson et al., 2007; Li et al., 2008; Liu & Howell, 2010), and chloroplast development (Kusnetsov et al., 1999; Miyoshi et al., 2003). These observations indicate that a variety of molecular functions are attributed to the NF-Y subunit family members in angiosperms, but no function has been assigned to those in P. patens.
In this report, we identified a new response element (ACTT) responsible for activating the ABI3-dependent ABA response pathway cooperatively with the nuclear factor Y (NF-Y) complex. Coexpression of PpABI3A along with PpNF-YC1 and PpNF-YB4 synergistically activated the ACTT-core element of the PpLEA1 promoter, but not the ACGT-core ABRE. The ability of NF-Y proteins to functionally enhance and support the expression of the ABI3-dependent ABA response pathway provides a framework for understanding the mechanism of how ABI3 interacts with transcriptional complexes to activate gene sets. These results further define the regulatory interactions at the transcriptional level for the expression of gene sets required for drought/desiccation tolerance and will shed light on the evolution of this pathway in the green plant lineage.