**4. Rice flowering regulation by Hd3a and GF14c interaction**

14-3-3 proteins in general bind to phosphoserine-modified proteins, as well as to some nonphosphorylated proteins such as exoenzyme S, which has no phosphorylated residue in its binding motif. They regulate the activities of a wide array of targets via direct protein-protein interactions, and effect changes in the client proteins. These changes can vary from inactivation or activation of the enzymatic activity of a target protein, to degradation or protection from degradation of the target, to movement of the target from one cellular location to another (usually nuclear-cytoplasmic shuttling). Plant 14-3-3 proteins bind a range of TFs and other signaling proteins, and have pivotal roles in regulating developmental and stress responses.

To reveal the biological functions of 14-3-3 in plants, the common approaches of generating overexpression, RNA suppression, or knockout plant lines probably have limited potential, for two reasons. First, because they interact with so many different targets, multiple pleiotropic effects would be expected. Second, 14-3-3s are encoded by a gene family with at least 12 expressed members in *Arabidopsis* [30], 8 members in rice [31, 32], and similar numbers in other plants, such as tomato and tobacco, which adds the complicating issues of isoform specificity and redundancy. However, working with a family of proteins that play so many roles will be interesting, given the challenge of trying to identify a particular protein-protein interaction associated with a phenotype using 14-3-3 mutants (overexpression or knockout mutants). Rather than focusing on 14-3-3s themselves, more targeted and informative approaches would be to identify specific interactions using biochemical or yeast two-hybrid methods, followed by *in vivo* confirmation and directed investigation of the potential 14-3-3 binding sites in target proteins.

Several studies of protein interactions involving FT/Hd3a orthologs have been published. In *Arabidopsis*, FT interacts with FD and 14-3-3 proteins [12, 13]. There are several SP interacting proteins in tomato, including a 14-3-3 family member, protein kinase and bZIP transcription factor [10]. SFT, another tomato ortholog of FT/Hd3a, also interacts with 14-3-3 as well as bZIP [11]. In rice, GF14c (G-box factor 14-3-3c protein) has been identified as a partner of Hd3a to activate particular gene in certain pathway. Since the 14-3-3 family members known to interact with various protein, the role of GF14c in Hd3a signaling is remarkable to elucidate. Functional analysis using knockout and overexpression approach were performed and the result indicate that *GF14c* is a negative regulator by interacting with *Hd3a* [33]. No diurnal changes or developmental patterns of *GF14c* expression were observed, indicating that *GF14c* is expressed independently of the photoperiod and abundantly throughout plant development.

#### **4.1. Subcellular localization of Hd3a and GF14c**

To identify the intracellular localization of Hd3a and GF14c, a fusion construct to express mCherry fluorescent protein-linked Hd3a under the ubiquitin promoter, and GFP-linked GF14c driven by the CaMV 35S promoter were made. These constructs were introduced into rice protoplasts. In all of the rice protoplasts observed in this experiment, Hd3a-mCherry localized in both cytoplasm and nucleus; however, GF14c-GFP was predominantly visualized in the cytoplasm (75% of rice protoplasts observed) (**Figure 8**). The predominant cytoplasmic localization of 14-3-3 proteins has led to the hypothesis that they might act as cytoplasmic anchors that either block import into the nucleus or other organelles, or promote export from organelles into the cytoplasm [34].

#### **4.2.** *In vitro* **and** *in vivo* **interaction of Hd3a and GF14c**

**4. Rice flowering regulation by Hd3a and GF14c interaction**

\* Length in amino acids identified from yeast two-hybrid screening.

**Table 2.** Proteins identified by yeast two-hybrid screening.

14-3-3 proteins in general bind to phosphoserine-modified proteins, as well as to some nonphosphorylated proteins such as exoenzyme S, which has no phosphorylated residue in its binding motif. They regulate the activities of a wide array of targets via direct protein-protein interactions, and effect changes in the client proteins. These changes can vary from inactivation or activation of the enzymatic activity of a target protein, to degradation or protection from degradation of the target, to movement of the target from one cellular location to another (usually nuclear-cytoplasmic shuttling). Plant 14-3-3 proteins bind a range of TFs and other signaling proteins, and have pivotal roles in regulating developmental and stress responses.

**Homologous proteins Accession number Two-hybrid\* Amino acids\*\* Domain/Function**

**115 256 14-3-3, adaptor,**

**147 361 GARP domain**

209 483 phosphatase

**– 112 366 TPX2 domain**

gi|50905035 121 141 –

gi|50726535 170 253 –

gi|27476086 157 657 chaperone

**chaperone, scaffolding**

Translation initiation factor 5A/eIF5A gi|50918889 161 161 – Linker histone gi|50921921 185 188 –

**AK122149/Os8g0430500**

Beta cyanoalanine synthase gi|55233175 377 377 –

AK100514/Os10g0203000

Putative SF16 protein gi|50931997 240 500 –

Unknown protein gi|50938743 68 68 – Unknown protein gi|54287657 91 91 –

Ser/Thr kinase gi|34894358 205 461 kinase Putative glutathione S-transferase gi|50900303 142 223 – Thioredoxin H-type (TRX-H) gi|50934769 204 350 –

**gi|50903393**

**Os02g0696900**

**Signaling**

58 Plant Engineering

**protein)**

**BIP116b**

(HSP70)

**Unknown**

**GF14c (G-box factor 14-3-3c** 

Putative anthranilate phosphoribosyltransferase

Putative NADPH-dependent retinol dehydrogenase/reductase

Putative heat shock 70 KD protein

\*\* Length in amino acids of protein.

**BRI1-KD interacting protein 116b/**

**OsKANADI1 gi|41053181**

Putative endonuclease/phosphatase gi|37532222

The interaction between Hd3a and GF14c was confirmed using several methods, including a GST pull-down assay, yeast two-hybrid and a co-immunoprecipitation [33]. A GST-Hd3a fusion protein was pulled down with His-tagged GF14c, indicating that Hd3a interacted with GF14c *in vitro*. Results of this experiment were thus consistent with the results of the yeast two-hybrid experiment. An *in vivo* interaction was also demonstrated by a co-immunoprecipitation experiment using rice suspension culture cells overexpressing myc-tagged Hd3a.

## **4.3. Bimolecular fluorescence complementation (BiFC) of Hd3a and GF14c**

To determine the distribution of Hd3a and GF14c *in vivo*, the BiFC technique was performed. The expression vector of Hd3a and GF14c fused to the N-terminal half of mVenus (Vn) and C-terminal half of mVenus (Vc), respectively. Those vectors were then transiently expressed on rice cell protoplasts. Plasmid mChery was used as a marker for transformed cells. The transformed rice protoplasts showed an interaction between Hd3a and GF14c as observed by strong green fluorescence that concentrated in the cytoplasm. The combination treatment of expression vectors using GUS to confirm the interaction cleary showed that the venus fluorescence only observed up to 10% of the transformed cells. This, indicated Hd3a only interact with GF14c mainly in cytoplasm [33].

#### **4.4. Confirmation of Hd3a interacting protein GF14c by mutant generation**

Several methods has been used to confirm the interaction between Hd3a and GF14c. All the methods used (yeast-two hybrid, in vitro pull-down, and BiFC assays) clearly showed that Hd3a and GF14c have strong interaction in rice. The subcellular distribution of Hd3a as well as GF14c demonstrated their localization in both the cytoplasm and nucleus.

GF14c acts as negative regulator of flowering and the overexpression of GF14c caused late flowering phenotype. Since the expression of Hd3a was observed both in the cytoplasm and nucleus, whereas GF14c is mainly in the cytoplasm, the possible interaction of these two protein is interesting to study. The mechanism of interaction of which GF14c would inhibit shuttling of Hd3a from the cytoplasm into the nucleus were confirmed by BiFC experiment. Based on data obtained it is clearly showed that Hd3a-GF14c is localized in cytoplasm. This indicate Hd3a-GF14c interaction lead to Hd3a cyoplasmic retention. The increasing of cytoplasmic retention of Hd3a can be explained as a model of late flowering by overexpression of GF14c. An example in *Arabidopsis*, the floral transition initiation in SAM, FT interacts with FD (bZIP transcription factor) that localized in the nucleus to induce the expression of target gene such as *AP1* [12]. In rice, this process could be attenuated by the cytoplasmic retention of Hd3a by GF14c. Several lines of evidence indicate a function for 14-3-3 proteins in nuclear-cytoplasmic shuttling in the signal transduction pathway [35, 36].

Another possible mechanism which can be explain the phenotype of GF14c overexpression and knockout mutant lines is the interaction between GF14c and Hd3a inhibit the movement of Hd3a from leaf to SAM. When GF14c expression level is low, Hd3a protein is capable to move along the floem to the SAM, since HD3a is small protein (about 20kDa) that is below of the size limit of plasmodesmata [37, 38]. The *gf14c* knockout mutant exhibited early flowering compare to WT. Remarkably, this mutant shown another phenotype such as dwarfism and increased tiller number. These pehotype suggesting that GF14c has another function independenlty with respect of flower induction at SAM.

According to Taoka [29] finding, 14-3-3 protein acts as intercellular receptor for Hd3a in the shoot apical cells. When Hd3a protein is transported to SAM, it will interact with 14-3-3 and form florigen activation complex (FAC). This FAC complex will be translocated into nucleus and binds to *OsFD1* (*FD* homolog of *A. thaliana*). The expression of *OsFD1* will lead to activation of flowering identity genes, resulting in the flowering. This finding helps us to understand the flowering mechanism in rice and also offers new approaches to manipulate the plants flowering in future.
