Interactions of B-class complex proteins involved in tepal development in Phalaenopsis orchid.
In our previous studies, we identified four DEFICIENS (DEF)-like genes and one GLOBOSA (GLO)-like gene involved in floral organ development in Phalaenopsis equestris. Revealing the DNA binding properties and protein-protein interactions of these floral homeotic MADS-box protein complexes (PeMADS) in orchids is crucial for the elucidation of the unique orchid floral morphogenesis. In this study, the interactome of B-class PeMADS proteins was assayed by the yeast two-hybrid system (Y2H) and glutathione S-transferase (GST) pull-down assays. Furthermore, the DNA binding activities of these proteins were assessed by using electrophoretic mobility shift assay (EMSA). All four DEF-like PeMADS proteins interacted individually with the GLO-like PeMADS6 in Y2H assay, yet with different strengths of interaction. Generally, the PeMADS3/PeMADS4 lineage interacted more strongly with PeMADS6 than the PeMADS2/PeMADS5 lineage did. In addition, independent homodimer formation for both PeMADS4 (DEF-like) and PeMADS6 (GLO-like) was detected. The protein-protein interactions between pairs of PeMADS proteins were further confirmed by using a GST pull-down assay. Furthermore, both the PeMADS4 homodimer and the PeMADS6 homodimer/homomultimer per se were able to bind to the MADS-box protein-binding motif CArG. The heterodimeric complexes PeMADS2-PeMADS6, PeMADS4-PeMADS6 and PeMADS5-PeMADS6 showed CArG binding activity. Taken together, these results suggest that various complexes formed among different combinations of the five B-class PeMADS proteins may increase the complexity of their regulatory functions and thus specify the molecular basis of whorl morphogenesis and combinatorial interactions of floral organ identity genes in orchids.
Tsai
Wen-Chieh
WC
Department of Biological Sciences and Technology, National Tainan University, Tainan 700, Taiwan.
Pan
Zhao-Jun
ZJ
Hsiao
Yu-Yun
YY
Jeng
Mei-Fen
MF
Wu
Ting-Feng
TF
Chen
Wen-Huei
WH
Chen
Hong-Hwa
HH
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D018744:DNA, Plant / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D035264:Flowers / Q000254:growth & development*; D026161:MADS Domain Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D029595:Orchidaceae / Q000254:growth & development*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques
D018744:DNA, Plant; D026161:MADS Domain Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins