Characterization of an AGAMOUS-like MADS Box Protein, a Probable Constituent of Flowering and Fruit Ripening Regulatory System in Banana

Swarup Roy Choudhury,Dibyendu N Sengupta,Sujit Roy,Anish Nag,Sanjay Kumar Singh,Stefan Kepinski

doi:10.1371/journal.pone.0044361

Abstract

The MADS-box family of genes has been shown to play a significant role in the development of reproductive organs, including dry and fleshy fruits. In this study, the molecular properties of an AGAMOUS like MADS box transcription factor in banana cultivar Giant governor (Musa sp, AAA group, subgroup Cavendish) has been elucidated. We have detected a CArG-box sequence binding AGAMOUS MADS-box protein in banana flower and fruit nuclear extracts in DNA-protein interaction assays. The protein fraction in the DNA-protein complex was analyzed by mass spectrometry and using this information we have obtained the full length cDNA of the corresponding protein. The deduced protein sequence showed ∼95% amino acid sequence homology with MA-MADS5, a MADS-box protein described previously from banana. We have characterized the domains of the identified AGAMOUS MADS-box protein involved in DNA binding and homodimer formation in vitro using full-length and truncated versions of affinity purified recombinant proteins. Furthermore, in order to gain insight about how DNA bending is achieved by this MADS-box factor, we performed circular permutation and phasing analysis using the wild type recombinant protein. The AGAMOUS MADS-box protein identified in this study has been found to predominantly accumulate in the climacteric fruit pulp and also in female flower ovary. In vivo and in vitro assays have revealed specific binding of the identified AGAMOUS MADS-box protein to CArG-box sequence in the promoters of major ripening genes in banana fruit. Overall, the expression patterns of this MADS-box protein in banana female flower ovary and during various phases of fruit ripening along with the interaction of the protein to the CArG-box sequence in the promoters of major ripening genes lead to interesting assumption about the possible involvement of this AGAMOUS MADS-box factor in banana fruit ripening and floral reproductive organ development.

Highlights

The MADS-box genes, which represent a highly conserved gene family of DNA-binding transcription factors, have been identified in a wide range of eukaryotic genomes including insects, amphibian, yeasts, mammals and plants [1]
Whereas 100 molar excess of unlabeled CArG box DNA clearly competed out the DNA binding activity (Figure 1b, lane 8 and 9), unlabeled GATA-box DNA was unable to compete the binding activity even at 100 molar excess ratio to labeled probe
Based on the observation of increased expression of MA-MADS5 transcripts in ripening banana fruit, we studied the accumulation levels of MA-MADS5 protein in banana fruit pulp during ex-planta ripening at different days after anthesis using affinity purified anti-MA-MADS5 polyclonal antibody (Text S2, Figure S7) We have observed an increased accumulation of MAMADS5 protein along with the ripening days at different days post anthesis (DPA) (Figure 5a, lanes 1–10)

Summary

Introduction

The MADS-box genes, which represent a highly conserved gene family of DNA-binding transcription factors, have been identified in a wide range of eukaryotic genomes including insects, amphibian, yeasts, mammals and plants [1]. The MADS box motif has been found as a typical and unique domain for the members of the MADS-box family of transcription factors which binds to a highly conserved DNA motif known as CArG box. Floral organ identity is controlled by diverse families of homeotic transcription factors. The floral homeotic gene AGAMOUS (AG) is a group C gene and it encodes a MADS box transcription factor [4]. AG interacts with other MADS box proteins to play essential function for the induction of reproductive organ development in Arabidopsis [5]

Methods

Results

Conclusion