Characterization of Three SEPALLATA-Like MADS-Box Genes Associated With Floral Development in Paphiopedilum henryanum (Orchidaceae)

Abstract

Paphiopedilum (Orchidaceae) is one of the world’s most popular orchids that is found in tropical and subtropical forests and has an enormous ornamental value. SEPALLATA-like (SEP-like) MADS-box genes are responsible for floral organ specification. In this study, three SEP-like MADS-box genes, PhSEP1, PhSEP2, and PhSEP3, were identified in Paphiopedilum henryanum. These genes were 732–916 bp, with conserved SEPI and SEPII motifs. Phylogenetic analysis revealed that PhSEP genes were evolutionarily closer to the core eudicot SEP3 lineage, whereas none of them belonged to core eudicot SEP1/2/4 clades. PhSEP genes displayed non-ubiquitous expression, which was detectable across all floral organs at all developmental stages of the flower buds. Furthermore, subcellular localization experiments revealed the localization of PhSEP proteins in the nucleus. Yeast two-hybrid assays revealed no self-activation of PhSEPs. The protein–protein interactions revealed that PhSEPs possibly interact with B-class DEFICIENS-like and E-class MADS-box proteins. Our study suggests that the three SEP-like genes may play key roles in flower development in P. henryanum, which will improve our understanding of the roles of the SEP-like MADS-box gene family and provide crucial insights into the mechanisms underlying floral development in orchids.

Functional conservation and divergence of SEPALLATA-like genes in floral development in Cymbidium sinense

“…Specifically, the mRNA of CsSEPs showed significant accumulation in flowers and pods, but was only mildly detected or even undetected in roots, stems, and leaves (Figures 3B-E). These results could be verified in other orchids, such as C. goeringii, E. pusilla, and P. henryanum, where the same expression pattern of SEP homologous genes is found to be expressed in floral organs, with low or no expression in vegetative organs (Lin et al, 2016;Dirks-Mulder et al, 2019;Sun et al, 2021;Cheng et al, 2022). However, in the flower bud development process (Figures 3F-I), the expression of CsSEP1 was significantly upregulated in S5.…”

“…This finding was consistent with previous reports on the phylogeny of SEP1/ 3-like genes in P. henryanum and P. equestris . In Orchids, four clades of SE P genes have been generated from orchid-specific duplications within monocot SEP clades ( Pan et al., 2014 ; Ai et al., 2021 ; Sun et al., 2021 ; Yang et al., 2021 ; Cheng et al., 2022 ). Therefore, we speculated that the endemic Orchid SEP1/2 and Orchid SEP3 branches were formed during plant evolution.…”

“…In Arabidopsis, B-class genes are direct targets of SEP proteins, and SEP3 displays extensive interactions with other MADS-box proteins ( de Folter et al., 2005 ). Similarly, in orchids, SEP-like proteins bind to CArG-box sequences and interact with AP1/FUL, B, C, D, and other MADS-box proteins during flower development ( Tilly et al., 1998 ; Pan et al., 2014 ; Mitoma and Kanno, 2018 ; Cheng et al., 2022 ). In the evolution of the orchid family, B-class MADS-box genes is diverged into different subclades, and involved with E-class SEP genes in the development of a highly modified perianth ( Pan et al., 2011 ; Lucibelli et al., 2021 ).…”

“…We retrieved partial SEP -like genes from previously published studies ( Yang et al., 2021 ; Cheng et al., 2022 ) and obtained others from the National Center for Biotechnology Information ( http://www.ncbi.nlm.nih.gov ) for phylogenetic analysis. The registration numbers for these genes are listed in Supplementary Table S2 .…”

“…For example, in P. equestris , the PeSEP3 gene can cause tepals to become leaf-like organs ( Pan et al., 2014 ). Additionally, the expression of SEP -like genes has been detected in all floral organs and plays a vital role in the development of the column, lip, and petals in Habenaria radiata and P. henryanum ( Mitoma and Kanno, 2018 ; Valoroso et al., 2019 ; Cheng et al., 2022 ). Defects in CeSEP1/3 -clade genes also contribute to the leaf-like flower phenotype in Cymbidium ensifolium mutants, consistent with research from Phalaenopsis ( Wei et al., 2020 ).…”