Expression and Functional Analyses of Nymphaea caerulea MADS-Box Genes Contribute to Clarify the Complex Flower Patterning of Water Lilies.

Silvia Moschin,Sebastiano Nigris,Giorgio Casadoro,Barbara Baldan,Enrico Cortese,Lucia Colombo,Simona Masiero,Stefano Cagnin,Ignacio Ezquer

doi:10.3389/fpls.2021.730270

Silvia Moschin, Sebastiano Nigris + Show 7 more

Open Access

https://doi.org/10.3389/fpls.2021.730270

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Abstract

Nymphaeaceae are early diverging angiosperms with large flowers characterized by showy petals and stamens not clearly whorled but presenting a gradual morphological transition from the outer elements to the inner stamens. Such flower structure makes these plant species relevant for studying flower evolution. MADS-domain transcription factors are crucial components of the molecular network that controls flower development. We therefore isolated and characterized MADS-box genes from the water lily Nymphaea caerulea. RNA-seq experiments on floral buds have been performed to obtain the transcript sequences of floral organ identity MADS-box genes. Maximum Likelihood phylogenetic analyses confirmed their belonging to specific MADS-box gene subfamilies. Their expression was quantified by RT-qPCR in all floral organs at two stages of development. Protein interactions among these transcription factors were investigated by yeast-two-hybrid assays. We found especially interesting the involvement of two different AGAMOUS-like genes (NycAG1 and NycAG2) in the water lily floral components. They were therefore functionally characterized by complementing Arabidopsis ag and shp1 shp2 mutants. The expression analysis of MADS-box genes across flower development in N. caerulea described a complex scenario made of numerous genes in numerous floral components. Their expression profiles in some cases were in line with what was expected from the ABC model of flower development and its extensions, while in other cases presented new and interesting gene expression patterns, as for instance the involvement of NycAGL6 and NycFL. Although sharing a high level of sequence similarity, the two AGAMOUS-like genes NycAG1 and NycAG2 could have undergone subfunctionalization or neofunctionalization, as only one of them could partially restore the euAG function in Arabidopsis ag-3 mutants. The hereby illustrated N. caerulea MADS-box gene expression pattern might mirror the morphological transition from the outer to the inner floral organs, and the presence of transition organs such as the petaloid stamens. This study is intended to broaden knowledge on the role and evolution of floral organ identity genes and the genetic mechanisms causing biodiversity in angiosperm flowers.

Highlights

The earliest angiosperm fossils date about 135 million years ago (MYA) (Willis and McElwain, 2013); it is increasingly recognized that angiosperms originated before (van der Kooi and Ollerton, 2020)
Overall our results indicate that NycAG1 can partially overcome AG absence regarding floral organ identity but it frequently not overcomes the floral meristem determination
This work provides an extensive and general view of the MADSbox genes expressed during flower development in N. caerulea, highlighting some peculiarities of the architecture of water lily flowers

Summary

Introduction

The earliest angiosperm fossils date about 135 million years ago (MYA) (Willis and McElwain, 2013); it is increasingly recognized that angiosperms originated before (van der Kooi and Ollerton, 2020). MADS-box genes are present in eukaryotes but are numerous in seed plants, where some gene-family members are especially important for the development of reproductive structures (Gramzow and Theißen, 2013) Detailed studies of their role have mostly been performed in eudicot model plant species, where functional genetics approaches have elucidated many of their gene functions. Based on data mostly from Arabidopsis, Antirrhinum, and Petunia, the genetic ABC model, later expanded into the ABCDE model, was proposed In this model, class A + E genes specify sepals, A + B + E genes specify petals, B + C + E specify stamens, C + E specify carpels and C + D + E specify ovules (Theiβen et al, 2016, and references therein). Studies in early diverging angiosperms (Buzgo et al, 2004; Kim et al, 2005; Soltis et al, 2007; Yoo et al, 2010) and in Ranunculales, a sister order to all other eudicots (Damerval and Becker, 2017 and references therein; Martínez-Gómez et al, 2021) evidenced both similarities and differences with respect to the ABCDE model

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