Characterization of NF–Y gene family and their expression and interaction analysis in Phalaenopsis orchid

The complex of Nuclear Factor Ys (NF-Ys), a family of heterotrimeric transcription factors composed of three unique subunits (NF-YA, NF-YB, and NF-YC), binds to the CCAAT box of eukaryotic promoters to activate or repress transcription of the downstream genes involved into various biological processes in plants. However, the systematic characterization of NF-Y gene family has not been elucidated in Phalaenopsis. A total of 24 NF-Y subunits (4 NF-YA, 9 NF-YB, and 11 NF-YC subunits) were identified in Phalaenopsis genome, whose exon/intron structures were highly differentiated among the PhNF-Y subunits. The distribution of motifs between coding regions of PhNF-YA and PhNF-YB/C was distinct. Segmental and tandem duplication events among paralogous PhNF-Ys were occurred. Six pairs of orthologous NF-Ys from Phalaenopsis and Arabidopsis and five pairs of orthologous NF-Ys from Phalaenopsis and rice involved in the phylogenetic gene synteny were identified. The various cis-elements being responsive to low-temperature, drought and ABA were distributed in the promoters of PhNF-Ys. qRT-PCR analysis indicated all of PhNF-Ys displayed the spatial specificity of expression in different tissues. Moreover, the expression levels of multiple PhNF-Ys significantly changed responding to low-temperature and ABA treatment. Yeast two hybrid and bimolecular fluorescence complementation assays approved the interaction of PhNF-YA1/3 with PhNF-YB6/PhNF-YC7, respectively, as well as PhNF-YB6 with PhNF-YC7. PhNF-YA1/3, PhNF-YB6, and PhNF-YC7 proteins were all localized in the nucleus. Further, transient overexpression of PhNF-YB6 and PhNF-YC7 promoted PhFT3 and repressed PhSVP expression in Phalaenopsis. These findings will facilitate to explore the role of PhNF-Ys in floral transition in Phalaenopsis orchid.