**1. Introduction**

Transition from vegetative phase to flowering involves many genetic pathways that interact with the external signals, such as day length and temperature, and internal signals such as hormones and developmental controls. One of the most important factors controlling flowering plants is response to daylight or photoperiod [1]. Based on photoperiodism, two

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

model plants, *Arabidopsis thaliana* and *Oryza sativa* are used to study on regulation of flowering time of long-day plant (LDP) and short-day plant (SDP), respectively. Three genes, which constitute a major genetic pathway in the photoperiodic regulation of flowering in rice, have recently been isolated. *O. sativa GIGANTEA* (*OsGI*), an ortholog of *Arabidopsis GI*, *Heading date 1* (*Hd1*), an ortholog of *Arabidopsis CO* (*CONSTANS*), and *Heading date 3a* (*Hd3a*), an ortholog of *FLOWERING LOCUS T* (*Arabidopsis FT*) are shown to form the main pathway for photoperiodic regulation of flowering in rice. These three genes were conserved between rice and *Arabidopsis*; however, the differences in their regulation results in either SDP or LDP (**Figure 1**). The major difference between rice, a SDP, and *Arabidopsis*, a LDP, was shown to be the regulation of *Hd3a/FT* by *Hd1/CO*. Under LD conditions, this regulation is positive in *Arabidopsis* while negative in rice [2].

In *Arabidopsis*, *GI* encodes a nuclear protein [3]. Expression of *GI* mRNA exhibits a circadian rhythm and *gi* mutants defects in clock function, indicating that *GI* is closely associated with the clock itself. *CO* encodes a transcription factor with B-box type zinc fingers thought to mediate protein-protein interaction. *FT* encodes a protein with homology to Raf kinase inhibitor protein (RKIP). It is a powerful promoter of flowering, activating the floral meristem identity gene, *APETALA1* (*AP1*) and is the target of several pathways controlling flowering time [4].

*Hd3a* was initially identified by quantitative trait locus (QTL) promoting flowering under short day-length condition. Using map-based cloning, *Hd3a* was determined as an ortholog of *FT* in *Arabidopsis* [5]. *Hd3a* overexpression under 35S promoter, native promoter, and vascular-specific promoters showed early flowering phenotype [6]. On the other hand, the suppression of *Hd3a* by RNA interference exhibited delayed flowering in rice [7]. These results strongly suggest that Hd3a protein function for flowering promotion.

**Figure 1.** Flowering pathway regulation in *Arabidopsis* and rice.

Hd3a shares sequence similarity with the mammalian phosphatidylethanolamine-binding protein (PEBP or RAF1 kinase inhibitor protein—RKIP) (**Figure 2**). The PEBP family regulates signaling pathways to control growth and differentiation. The PEBPs seem to act biochemically as inhibitors, binding signaling components to modulate the flux through their pathways.

model plants, *Arabidopsis thaliana* and *Oryza sativa* are used to study on regulation of flowering time of long-day plant (LDP) and short-day plant (SDP), respectively. Three genes, which constitute a major genetic pathway in the photoperiodic regulation of flowering in rice, have recently been isolated. *O. sativa GIGANTEA* (*OsGI*), an ortholog of *Arabidopsis GI*, *Heading date 1* (*Hd1*), an ortholog of *Arabidopsis CO* (*CONSTANS*), and *Heading date 3a* (*Hd3a*), an ortholog of *FLOWERING LOCUS T* (*Arabidopsis FT*) are shown to form the main pathway for photoperiodic regulation of flowering in rice. These three genes were conserved between rice and *Arabidopsis*; however, the differences in their regulation results in either SDP or LDP (**Figure 1**). The major difference between rice, a SDP, and *Arabidopsis*, a LDP, was shown to be the regulation of *Hd3a/FT* by *Hd1/CO*. Under LD conditions, this regulation is positive in

In *Arabidopsis*, *GI* encodes a nuclear protein [3]. Expression of *GI* mRNA exhibits a circadian rhythm and *gi* mutants defects in clock function, indicating that *GI* is closely associated with the clock itself. *CO* encodes a transcription factor with B-box type zinc fingers thought to mediate protein-protein interaction. *FT* encodes a protein with homology to Raf kinase inhibitor protein (RKIP). It is a powerful promoter of flowering, activating the floral meristem identity gene, *APETALA1* (*AP1*) and is the target of several pathways controlling flowering

*Hd3a* was initially identified by quantitative trait locus (QTL) promoting flowering under short day-length condition. Using map-based cloning, *Hd3a* was determined as an ortholog of *FT* in *Arabidopsis* [5]. *Hd3a* overexpression under 35S promoter, native promoter, and vascular-specific promoters showed early flowering phenotype [6]. On the other hand, the suppression of *Hd3a* by RNA interference exhibited delayed flowering in rice [7]. These

results strongly suggest that Hd3a protein function for flowering promotion.

**Figure 1.** Flowering pathway regulation in *Arabidopsis* and rice.

*Arabidopsis* while negative in rice [2].

time [4].

50 Plant Engineering

The crystal structure of PEBP from human and bovine sources, CEN protein from *Antirrhinum* [8] and that of the Terminal Flowering Locus (TFL) and FT proteins from *Arabidopsis* [9] have been determined. In **Figure 3**, the molecular model of Hd3a protein was built using the Swiss-Prot automated comparative protein modeling server, based on its sequence homology to two members of the RKIP protein family whose structures have been determined by X-ray crystallographic methods (*Arabidopsis* FT and TFL1, protein databank accession numbers 1WKP and 1WKO, respectively). Structural analysis of these proteins indicated the accessibility of the ligand-binding pocket to interact with the proteins partner.

PEBPs might also act as either scaffolds for or regulators of signaling complexes, as showed by the finding that Self-Pruning (SP) and Single-Flower Truss (SFT), as a tomato homolog of Terminal Flowering Locus 1 (TFL1) and Flowering Locus T (FT), respectively, can interact with a range of diverse proteins [10, 11]. Several studies of protein interactions involving FT/Hd3a orthologs have been published. In *Arabidopsis*, FT interacts with the basic/leucine zipper (bZIP) transcriptional factor FD [12, 13]. Interestingly, the bZIP transcription factor Self-pruning G-box protein (SPGB) in tomato, a homolog of FD, interact with SP and SFT as well. Moreover, FT interacts with FD and 14-3-3 proteins (**Table 1**) [11–13]. However, no Hd3a interacting proteins have yet been identified in rice.

**Figure 2.** Hd3a shares high homology with other phosphatidylethanolamine binding protein or Raf kinase inhibitor protein (PEBP/RKIP) in various plant species. Hd3a and FT has 73% homology. The blue box indicates the amino acid difference which is responsible for its function as flower inducer (tyrosin in FT and Hd3a) or repressor (histidine in CEN, SP, and TFL1).

**Figure 3.** Molecular model of Hd3a. Hd3a protein contains a large central β-sheet (yellow ribbons), which is flanked on one side by a smaller β-sheet and on the other by an α-helix (red ribbons).

In *Arabidopsis*, it is believed that the combination of interacting proteins, resolved crystal structures, and mutant phenotype analysis will lead to a comprehensive understanding of the mechanisms that facilitate the switch from vegetative phase to reproductive phase. It seems likely that Hd3a/FT is involved not only in flowering, but also in other aspects of growth and development in plant architectures [11, 15–18]. This function will be achieved by interacting with its partners. Hd3a might recruit different proteins to perform its roles in plant growth and development, particularly during floral transition.

In this chapter, we will discuss the regulation of Hd3a florigen in rice and the identification of novel interaction partners for rice Hd3a protein using yeast two-hybrid screening. The interaction between Hd3a and its partners was further confirmed by several methods, such as yeast two-hybrid assay using full-length cDNA, *in vitro* pull-down assay, co-immunoprecipitation, and bimolecular fluorescence complementation (BiFC). The expression pattern and subcellular localization of each Hd3a interacting partner provided important insights into its function. To further characterize the function of Hd3a interacting proteins in plant growth


**Table 1.** PEBP/RKIPs interacting proteins.

and development, particularly during the floral transition, a reverse genetics approach was used to generate gain of function mutants (overexpression)/knockdown mutants using RNAi or knockout mutants.
