**5. Hd3a interaction with BIP116b and OsKANADI**

Analysis of Hd3a interacting proteins in yeast two-hybrid screening identified a novel protein homolog of BIP116 (brassinosteroid-insensitive 1 [BRI1] kinase domain interacting protein 116) and a novel putative transcription factor belonging to the KANADI domain protein family, namely OsKANADI1 (OsKAN1).

### **5.1. Hd3a interact with BIP116b**

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

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

as GF14c demonstrated their localization in both the cytoplasm and nucleus.

shuttling in the signal transduction pathway [35, 36].

respect of flower induction at SAM.

with GF14c mainly in cytoplasm [33].

60 Plant Engineering

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

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

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

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

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 BRI1 has an extracellular domain (containing an N-terminal signal peptide, and LRR-RK and island domains), a transmembrane domain, a kinase domain and a C-terminal peptide [39]. Only a partial C-terminal region fragment was identified initially from yeast two-hybrid screening. An experiment using full-length Hd3a and full-length BIP116b, either as bait or as prey, further confirmed that Hd3a and BIP116b interact in the yeast system. To further study the interaction between Hd3a and BIP116b *in vitro*, a GST pull-down assay was performed. A GST-BIP116b fusion protein was pulled down with His-tagged Hd3a. However, an interaction could not be found between Hd3a and BIP116b, even though both proteins were expressed in *Escherichia coli*. One possible explanation for this discrepancy is that posttranslational modification of BIP116b may be necessary for binding.

The transgenic plant for either overexpression or RNAi mutant were generated. The results showed that the BIP116ab RNAi plants exhibited delayed flowering compared to wild-type plant, and the overexpressed plant showed branching and increasing tiller number similar to Hd3a overexpressed plants. Therefore, further characterization in mutant plants will be interesting to get an insight into its function.

#### **5.2. Hd3a interact with OsKANADI1**

In *Arabidopsis*, *KANADI* genes function in lateral polarity in organs including roots, leaves, and flowers. *KANADI* is required for abaxial identity in both leaves and carpels. It encodes a nuclear-localized protein in the GARP family of putative transcription factors [40]. GARP homologs constitute a large family of DNA-binding proteins in plants that may be needed for a variety of key cellular functions including regulation of transcription, phosphor transfer signaling, and differentiation. A GARP motif was also found in the identified *KANADI* product [41].

*KANADI* acts antagonistically to the *class III HZ-Zip* genes. *KANADI* genes are expressed in a pattern complementary to that of the *class III HD-Zip* genes in the shoot; *KANADI* expression occurs in the phloem and abaxial regions of lateral organs early in development [41–43]. While *KANADI* genes do not appear to be required for proper meristem function, they are needed for pattern formation of organs produced by the shoot apical and vascular meristems.

Sequence analysis showed that OsKAN1 consists of six exons. Phylogenetic and comparative genetic analysis indicated that OsKAN1 is an ortholog of AtKANs and is most closely related to AtKAN4. The predicted OsKAN1 polypeptide contains a potential phosphorylation site in the C-terminal region which may be important for interaction with other proteins involved in signaling. The overall identity between *KANADI* family members is low, but they are nearly identical within the GARP domain. The sequence similarity found in this subset of GARP genes may indicate that they have overlapping or partially redundant functions.

Suppression of OsKANADI1 by RNAi displayed branching and increasing tiller number in several lines [44]. This phenotype resembles to the Hd3a overexpressed plants indicating that they possibly function in similar pathway. Further characterization using the OsKANADI mutants plants in the next generation remains to be investigated.
