*3.3. Subcellular Localization of PlNCEDs*

Protein maintains its optimal function in a specific subcellular localization. Therefore, to unravel the cellular functions of *PlNCEDs* during seed dormancy and its release process, we also carried out fluorophore tagging of the protein using green fluorescent protein to locate the presence of *PlNCED* proteins within the cell. Fluorescence microscopic analysis showed that the GFP fluorescence signal was distributed in the cell membrane, nucleus, and cytoplasm of *A. thaliana* protoplast containing the empty 16318-hGFP vector (Figure 3). In contrast, the 16318-hGFP-PlNCED1 and 16318-hGFP-PlNCED2 fusion proteins were

only observed in the nucleus and cytoplasm, respectively (Figure 3). This differential cellular localization of two PlNCEDs implies that PlNCED1 may play a genetic role similar to that of transcription factors, whereas PlNCED2 may act as a functional enzyme to synthesize ABA.

**Figure 3.** Subcellular localization of the 16318-hGFP-PlNCED fusion protein in protoplast.
