Interactions of B-class complex proteins involved in tepal development in Phalaenopsis orchid

Abstract

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.

The genome sequence of the orchid Phalaenopsis equestris

“…Genes in the B-class AP3, C/D-class and E-class clades are well known for their roles in the specification of floral organ identity and have been well studied in P. equestris. These expanded clades including members with differential expression patterns in orchid floral organs, as well as divergent encoded protein domains, support the unique evolutionary routes of these floral organ identity genes associated with the unique labellum and gynostemium innovation in orchids [47][48][49][50] . Notably, one of the three gene copies in the expanded AGL6 clade had an expression pattern similar to that of the AP3-like PeMADS4 gene, which is specifically expressed in the labellum 51 .…”

The Tarenaya hassleriana Genome Provides Insight into Reproductive Trait and Genome Evolution of Crucifers

“…Both PI and AP3 in Tarenaya have diverged in expression levels. MADS box B-class gene homologs in the AP3 lineage as well as TCP members have been implicated in the establishment of monosymmetric flowers in monocots, including Orchidaceae species (Tsai et al, 2004(Tsai et al, , 2008Mondragón-Palomino and Theissen, 2009;Bartlett and Specht, 2010;Preston and Hileman, 2012), PI genes have contributed to floral diversification in Asterids (Viaene et al, 2009), and B-class genes have contributed to Aquilegia floral diversification (Kramer et al, 2007). Thus, it is possible that both B-class and TCP genes have an impact on floral monosymmetry in Cleomaceae.…”

TheseirenaB Class Floral Homeotic Mutant of California Poppy (Eschscholzia californica) Reveals a Function of the Enigmatic PI Motif in the Formation of Specific Multimeric MADS Domain Protein Complexes

“…The obligate B protein heterodimers in snapdragon and Arabidopsis are required both for organ identity specification and to maintain B gene expression in an autoregulatory circuit Tröbner et al, 1992;Zachgo et al, 1995;Davies et al, 1996;Hill et al, 1998;Tilly et al, 1998;Manchado-Rojo et al, 2012). In addition to heterodimers of AP3/PI-like proteins, homodimers of AP3 orthologs have been found in basal eudicots like columbine (Aquilegia vulgaris) and opium poppy (Papaver somniferum) (Drea et al, 2007;Kramer et al, 2007), whereas homodimer formation of PI orthologs has been demonstrated so far for petaloid monocots like the tulip (Tulipa gesneriana) and the orchid Phalaenopsis equestris (Kanno et al, 2003;Tsai et al, 2008). However, a function could not be assigned to homodimers formed by either AP3 or PI orthologs.…”