**2. Flowering regulation in rice**

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

**Figure 3.** Molecular model of Hd3a. Hd3a protein contains a large central β-sheet (yellow ribbons), which is flanked on

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

**Protein Interacting proteins References** Mammalian PEBP Raf1 [14]

Flowering Locus T (FT) *Arabidopsis thaliana* FD (bZIP transcription factor) [12, 13]

14-3-3/74

SPGB (a putative bZIP transcription factor)

[10]

[11]

SPAK (SP-associated kinase)

and development, particularly during floral transition.

Self-Pruning (SP) tomato 14-3-3 (adapter protein)

Single-Flower Truss (SFT) tomato SPGB

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

one side by a smaller β-sheet and on the other by an α-helix (red ribbons).

52 Plant Engineering

In rice (*O. sativa* L.), flowering is mainly induced by photoperiod. *GIGANTEA* in rice (*OsGI*) is a clock-regulated gene, which was first identified by the differential display method, and its expression is high in the middle of day [2]. Under inductive SD conditions, *OsGI* promotes the expression of *Heading date 1* (*Hd1*), and *Hd1* activates *Hd3a* expression. However, under noninductive LD conditions, *Hd1* suppresses *Hd3a* expression [2]. *OsGI* acts as the primary upstream regulator of *Hd1* expression [19]. OsGI is a large protein that is present in both the nucleus and cytoplasm of rice cells [12]. Suppression of *OsGI* by RNAi or antisense expression caused late flowering and reduced *Hd1* transcription under SD conditions [2].

Several unique genes in rice were isolated. *Early heading date1* (*Ehd1*) encodes a B-type response regulator and is a unique flowering time gene [20]. *Ehd1* promotes floral transition preferentially under SD conditions, even in the absence of functional alleles of *Hd1*. Expression analysis revealed that *Ehd1* functions upstream of *Hd3a*, *RFT1*, and some MADS-box genes [20]. More recently, *Ghd7* (for grain number, plant height, and heading date 7), which encodes a *CO*, *CO-LIKE*, and *TIMING OF CAB1* (*CCT*)-domain protein, was isolated from natural variants in rice [21]. *Ghd7* affects transcript levels of *Ehd1* and *Hd3a* under LD conditions and delayed the flowering. In the same condition, *Ghd7* does not affect *Hd1*. Therefore, rice have different flowering pathway: *Hd1* pathway and *Ehd1* pathway. These two different pathway integrated with environmental condition, in this case is photoperiod and regulate the FT-like genes to initiate or delay the flowering [22]. Another unique flowering gene in rice, *RID*/*OsId1*/*ehd2*, yields an extremely late flowering phenotype under both SD and LD conditions. This gene encodes a putative transcription factor with a zinc finger motif, and is an ortholog of *INDETERMINATE1* (*ID1*), which promotes flowering in maize (*Zea mays*). Specifically, it promotes the floral transition, mainly by upregulating *Ehd1* and genes downstream of *Ehd1*, such as *Hd3a* and *RFT1* [23–25].

*Hd3a* expression under SD conditions is also regulated by phytochrome. In the *se5* (the photoperiodic sensitivity 5) mutant, which lacks a functional gene encoding heme oxygenase [26], an enzyme that is required for loss-of-function alleles in one of the three rice phytochromes [27], rice exhibited early in flowering. *Hd1* expression is not affected by *se5* or *phyB* mutations; thus, phytochrome represses *Hd3a* expression downstream or independently of *Hd1* expression under SD conditions.

During the vegetative phase in rice, the shoot apical meristem (SAM) produces a series of leaves. The vegetative parts of the rice plant, consisting of root, culm, and leaves, form a tiller. A dramatic change occurs during the transition from vegetative to reproductive stages, with the tiller terminating to produce leaf and the panicle (inflorescence) being generated on the uppermost internode of the culm (**Figure 4**). The development stage of the young panicle is also related to the timing of leaf emergence. The differentiation stage of the young panicle almost

**Figure 4.** Rice plant during the developmental stages: vegetative phase, early reproductive phase (left panel) and during seed maturation (right panel).

directly correlates with the start of emergence of the fourth leaf (counted downward from the flag leaf). At the time of flag leaf (small last leaf) emergence, the glumes flower primordial has already differentiated and pollen mother cells are being formed [28]. The flag leaf, contributes largely to the filling of grains because it supplies photosynthetic products mainly to the panicle.

Flowering time in rice is indicated by the emergence of the flag leaf or the panicle (heading date). The panicle is initiated when the first bract primordium differentiates on the shoot apex, approximately 30 days before panicle emergence (heading). The start of differentiation of the glumes flower primordial follows after the rachis-branches differentiation has occurred (24 days before heading). The pistil and stamen differentiate 20 days before heading. Meiosis in the anther occurs 12 days before heading, and flower organ completion occurs 1–2 days before heading.
