| Summary: | Common buckwheat (<i>Fagopyrum esculentum</i>) produces distylous flowers with undifferentiated petaloid tepals, which makes it obviously different from flowers of model species. In model species <i>Arabidopsis</i>, <i>APETALA3</i> (<i>AP3</i>) is expressed in petal and stamen and specifies petal and stamen identities during flower development. Combining with our previous studies, we found that small-scale gene duplication (GD) event and alternative splicing (AS) of common buckwheat <i>AP3</i> orthologs resulted in <i>FaesAP3_1</i>, <i>FaesAP3_2</i> and <i>FaesAP3_2a</i>. <i>FaesAP3_2</i> and <i>FaesAP3_2a</i> were mainly expressed in the stamen of thrum and pin flower. Promoters functional analysis suggested that intense GUS staining was observed in the whole stamen in <i>pFaesAP3_2::GUS</i> transgenic <i>Arabidopsis</i>, while intense GUS staining was observed only in the filament of stamen in <i>pFaesAP3_1::GUS</i> transgenic <i>Arabidopsis.</i> These suggested that <i>FaesAP3_1</i> and <i>FaesAP3_2</i> had overlapping functions in specifying stamen filament identity and work together to determine normal stamen development. Additionally, <i>FaesAP3_2</i> and <i>FaesAP3_2a</i> owned the similar ability to rescue stamen development of <i>Arabidopsis ap3-3</i> mutant, although AS resulted in a frameshift mutation and consequent omission of the complete PI-derived motif and euAP3 motif of <i>FaesAP3_2a</i>. These suggested that the MIK region of <i>AP3</i>-like proteins was crucial for determining stamen identity, while the function of <i>AP3</i>-like proteins in specifying petal identity was gradually obtained after <i>AP3</i> Orthologs acquiring a novel C-terminal euAP3 motif during the evolution of core eudicots. Our results also provide a clue to understanding the early evolution of the functional specificity of euAP3-type proteins involving in floral organ development in core eudicots, and also suggested that <i>FaesAP3_2</i> holds the potential application for biotechnical engineering to develop a sterile male line of <i>F. esculentum.</i>
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