The Greenish Flower Phenotype of Habenaria radiata (Orchidaceae) Is Caused by a Mutation in the SEPALLATA-Like MADS-Box Gene HrSEP-1.

Mai Mitoma,Akira Kanno

doi:10.3389/fpls.2018.00831

Abstract

In Arabidopsis thaliana, the E-class SEPALLATA (SEP) genes are generally expressed across all floral whorls. These genes play fundamental roles in floral organ fate determination during development by interacting with other MADS-box gene products, such as those from A-, B-, and C-class genes. However, the function of SEP genes in orchid remains obscure. Here, we analyzed a mutant orchid cultivar with greenish flowers in Habenaria radiata and found that this phenotype is caused by the absence of SEP function. Wild type H. radiata flowers contain a column and two perianth whorls consisting of three greenish sepals, two white petals, and a lip (labellum). By contrast, the flowers of H. radiata cultivar ‘Ryokusei’ appear greenish, with three normal sepals in whorl 1, two greenish petals and a lip in whorl 2, and several sepaloid organs and a ventral column in whorls 3 and 4. We isolated two SEP-like genes (HrSEP-1 and HrSEP-2) and two AGAMOUS-like genes (HrAG-1 and HrAG-2) from wild type H. radiata and compared their expression in the wild type vs. the mutant cultivar. HrAG-1 and HrAG-2 were expressed in the column in the wild type, whereas these genes were expressed in the ventral column and in sepaloid organs that had been converted from a column in ‘Ryokusei.’ HrSEP-1 and HrSEP-2 were expressed in all floral organs in the wild type. However, in the mutant cultivar, HrSEP-2 was expressed in all floral organs, while HrSEP-1 expression was not detected. Thus, we analyzed the genomic structures of HrSEP-1 in the wild type and ‘Ryokusei’ and identified a retrotransposon-like element in its first exon in ‘Ryokusei.’ Yeast two-hybrid assays demonstrated that HrSEP-1 interacts with HrDEF, HrAG-1, and HrAG-2. These results indicate that the mutant phenotype of ‘Ryokusei’ flowers is caused by the loss of function of HrSEP-1. Therefore, this gene plays an important role in column, lip, and petal development in H. radiata flowers.

Highlights

The ABC model of floral organ identity determination was established based on genetic studies in Arabidopsis thaliana and Antirrhinum majus (Carpenter and Coen, 1990; Bowman et al, 1991; Schwarz-Sommer et al, 1992)
Since the floral morphology of the wild type and ‘Ryokusei’ is very different, we analyzed the sequences of the nuclear ribosomal internal transcribed spacer (ITS) regions from ‘Ryokusei’ and various Habenaria species to confirm the origin of this mutant cultivar (Supplementary Figure S1)
A Retrotransposon Insertion Is Present in HrSEP-1 in ‘Ryokusei’

Summary

Introduction

The ABC model of floral organ identity determination was established based on genetic studies in Arabidopsis thaliana and Antirrhinum majus (Carpenter and Coen, 1990; Bowman et al, 1991; Schwarz-Sommer et al, 1992). According to this model, the activity of A-, B-, and C-class genes, alone or in combination, specifies the formation of the distinct organs of the four floral. The A-class MADS-box gene APETALA1 (AP1)-like is required for the establishment of floral meristem and for specifying sepal and petal identity. The C-class AGAMOUS (AG) genes play a central role in stamen and carpel development (Yanofsky et al, 1990)

Methods

Results

Conclusion