Background

2.1.1 FT AND TFL1 REGULATION OF DOWNSTREAM FLOWERING GENES

Several interconnected pathways control flowering in Arabidopsis. These pathways form a gene network that responds to environmental input to induce flowering. The photoperiod-regulated flowering pathway relies on day length to control flowering (Andrés and Coupland, 2012). Two particularly important genes in this pathway are FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1). FT and TFL1 encode transcriptional regulators with homology to phosphatidylethanolamine- binding proteins (PEBPs) (Hanzawa et al., 2005). Diverse proteins found in bacteria, animals and plants, PEBPs function in signaling pathways involved in growth and differentiation (Hanzawa et al., 2005; Karlgren et al., 2011). The transcriptional regulators encoded by FT and TFL1 share ~60% amino acid sequence identity but function in an opposite manner (Hanzawa et al., 2005; Hanano and Goto, 2011). FT promotes the transition to reproductive development and flowering, while TFL1 represses this transition (Hanzawa et al., 2005; Hanano and Goto, 2011). Their high sequence homology suggests conserved biochemical action, but much remains unknown about the mechanisms by which FT and TFL1 control downstream flowering gene expression (Hanzawa et al., 2005; Hanano and Goto, 2011).

FT expression is induced by the circadian clock-controlled transcription factor CONSTANS under flowering-inductive long-day conditions (Wigge, 2011; Andrés and Coupland, 2012). FT protein moves from the leaves through the phloem until it reaches the shoot apical meristem (Takada and Goto, 2003; Imaizumi and Kay, 2006; Corbesier et al., 2007; Lin et al., 2007; Wigge, 2011). After reaching the meristem, FT binds to the transcription factor FLOWERING LOCUS D (FD) to form a complex that regulates meristem identity genes, resulting in stimulation of flowering (Abe et al., 2005; Hanano

SQUAMOSA BINDING LIKE (SPL) transcription factors, which promote the transcription of meristem identity genes such as LEAFY (LFY), APETALA1 (AP1) and FRUITFULL (FUL) (Wang et al., 2009; Yamaguchi et al., 2009; Andrés and Coupland, 2012; Jung et al., 2012). The actions of meristem identity genes reprogram the primordia to produce reproductive structures instead of vegetative ones (Blázquez et al., 2006; Amasino, 2010).

TFL1 promotes inflorescence meristem identity and suppresses the transition to the reproductive phase by acting at the shoot apical meristem (Ratcliffe et al., 1998; Liljegren et al., 1999; Blázquez et al., 2006; Benlloch et al., 2007). At the molecular level, TFL1 operates by preventing meristem identity genes (such as LFY and AP1) from acting at the center of the shoot apex (Ratcliffe et al., 1999). It both inhibits these floral meristem genes’ expression and prevents the meristem from responding to them (Ratcliffe et al., 1999). However, the molecular mechanism by which TFL1 modulates downstream genes remains largely unknown. TFL1 may suppress LFY, AP1 and other meristem identity genes by partnering with FD, with which it weakly interacts (Abe et al., 2005; Hanano and Goto, 2011). When fused to the transcriptional activator VP16 in wild-type Arabidopsis, TFL1 induces the expression of a number of meristem identity genes, including LFY and AP1 (Hanano and Goto, 2011). Therefore, LFY and AP1 are targets of TFL1 (Hanano and Goto, 2011). However, whether TFL1 directly or indirectly controls expression of these genes remains unclear (Hanano and Goto, 2011).

2.1.2 INDUCTION SYSTEMS

A number of systems to induce gene expression have been developed for use in plants. As opposed to constitutive promoters, which express a target gene at high levels throughout development, chemical-inducible systems allow direct control over a particular gene at a particular point in development (Zuo et al., 2000a). Such systems remain inactive until induced by application of a chemical (Zuo et al., 2000a). Induction systems using steroid hormones (such as dexamethasone and estradiol), ethanol, and heat shock have enjoyed widespread use (Aoyama and Chua, 1997; Matsuhara et al., 2000; Zuo et al., 2000a; Borghi, 2010). These systems generally consist of two separate transcription units. The first unit contains a constitutive promoter (such as 35S) for high expression of a chemically responsive transcription factor (Zuo et al., 2000a; Borghi,

2010). The second unit contains several copies of the transcription factor binding site connected to a minimal promoter (typically 35S) for constitutive expression of the target gene (Zuo et al., 2000a; Borghi, 2010). Application of the chemical inducer may occur by a variety of routes, such as by spraying plants with the chemical or by adding the chemical to soil (Borghi, 2010).

In the glucocorticoid induction system used in this project, the transformation vector pGREEN-0229-35S:GR (pGreen) contains the T-DNA region that is incorporated into the plant genome by Agrobacterium tumefaciens (Agrobacteria)-mediated transformation (Hellens et al., 2000; Yu et al., 2004). In the T-DNA transcription unit containing the target gene, the Cauliflower Mosaic Virus 35S promoter and the hormone-binding domain of the rat glucocorticoid receptor flank opposite ends of the target gene cloning site (Yu et al., 2004). The target gene and glucocorticoid receptor domain are transcribed and translated as a chimeric protein. The helper plasmid pSoup carries the replicase gene required for pGreen to replicate in Agrobacteria (Hellens et al., 2000). This induction system uses post-translational induction. After translation, the chimeric protein consisting of the target protein fused to the glucocorticoid receptor domain remains in the cytoplasm because it associates with regulatory proteins such as Hsp90 (Zuo et al., 2000a; Borghi, 2010). Application of the glucocorticoid hormone dexamethasone disrupts this association and causes the fusion protein to dimerize and translocate to the nucleus, where it affects gene expression (Zuo et al., 2000a; Borghi, 2010). Pitfalls of this system include the large size of the glucocorticoid receptor domain, which could impact native protein function; dexamethasone toxicity on plant tissue; and dexamethasone-induced activation of defense-related genes (Zuo et al., 2000a).

In the estradiol induction system used in this project, the transformation vector pER8 contains the T-DNA region that is incorporated into the plant genome by Agrobacterium-mediated transformation (Zuo et al., 2000b). In the T-DNA transcription unit containing the target gene, the 35S minimal promoter precedes the insertion site to incite high levels of target gene expression (Zuo et al., 2000b). Unlike the glucocorticoid induction system, this system uses transcriptional induction, in which application of the hormone estradiol directly activates expression of a target gene.

Estradiol also differs from dexamethasone in that it appears not to activate defense- related genes or cause toxicity to plant tissue (Zuo et al., 2000b).

2.1.3 EXPERIMENTAL HYPOTHESIS AND AIMS

We hypothesized that FT and TFL1 modulate the same genetic pathways because they regulate flowering in an opposite manner but possess high amino acid sequence homolog and both interact with FD. We sought to investigate which downstream genes TFL1 regulates and whether FT and TFL1 directly regulate the same set of genes. In order to answer these questions, we followed two approaches. The first approach used RNA sequencing to examine gene expression in both TFL1 overexpression and loss-of- function plants. The second approach used two induction systems to identify the immediate targets of FT and TFL1.