Studies On The Regulatory Mechanism Of Floral Organ Petaloid In Lotus(Nelumbo Nucifera)

Lotus,a basal eudicot in the family of Nelumbonaceae,is a perennial aquatic plant,comprising of two species,Nelumbo nucifera Gaertn.and Nelumbo lutea Pers.China is one of the centers of lotus distribution and cultivation.As a famous traditional flower in China,the lotus is a vital ornamental aquatic plant,whose ornamental value largely depends on its gorgeous colors and varied flower shapes.The flower shapes include the number of the petal,petal size,and petal shape etc.The petal-like feature is one of breeding selection for ornamental plant.Several lotus cultivars form petaloid feature during flower development.Double flower of lotus is majorly constituted by stamen petaloid and carpel petaloid Up to now,the molecular mechanisms of stamen petaloid and carpel petaloid in lotus remain unclear.In this study,through omics(transcriptomic,proteomic,and genome-wide DNA methylation)associated analysis to explore the candidate petaloid genes,a basic theory of petaloid mechanism in lotus was provided.Molecular biotechnology techniques can be applied for improvement of lotus morphology,effectively enriching new cultivars in flower lotus.Therefore,it is very significant for breeding the novel variety of ornamental flower lotus that explores excellent genes involved in the flower shape.MADS-box gene family was identified and analyzed,and their expression patterns in various tissues examined.35S::NnSEP3 transgenic line showed affected flowering time and flower morphology in Arabidopsis thaliana.The main results are summarized as follows:This study covers the comprehensive analysis of transcriptomic data to provide an overview of the transcriptional changes in different floral organs in lotus cultivar'Fenhonglingxiao'.In addition,through pairwise comparison analyses between three floral organs,we identified 3900 DEGs likely involved in floral organ identity.Outstandingly,the main eleven hormone-related genes and 22 TFs are involved in the conversion of the stamen to stamen-petaloid.We report that 29 DEGs were detected in floral organs and confirmed by qRT-PCR,and shows significant accordance with RNA-seq result.Additionally,the expression pattern of fifteen candidate genes(including seven MADS-box genes)was evaluated in eight different tissues by qRT-PCR.We hypothesize that their function together in converting stamen to stamen petaloid.We identified some MADS-box genes being potential regulators of ambiguous boundaries of petal and stamen.The DNA methylation profiles in the petal,stamen petaloid,and stamen of lotus provided clues for further understanding of the genetic pathway regulatory roles in floral organs formation.The methylation level of different floral organs exhibited significant changes in CHH context.In addition,the methylation level in introns was higher compared to exons.Under DNA methylation association with RNA-Seq data,31 DMR-associated genes were identified.In particular,a U-box domain-containing protein 33 encoding gene might play crucial roles in floral organ formation.We combined transcriptomic and proteomic analysis to the overview of the transcriptional changes and the expression of proteins changes in carpel,carpel petaloid,petal,stamen petaloid,and stamen of lotus cultivar 'Sleeping beauty'.Several transcription factors were involved in petaloid.Especially,one member of MADS-box genes,homeotic gene AG plays a critical role in flower development.A total of 44 MADS-box family genes were genome-wide identified in the whole lotus genome in the current study.This study is a systematic report to investigate the MADS-box gene family by characterizing the phylogenetic topology relationships,gene structures,conserved domain and motif,and spatial expression patterns of its members in sacred lotus.35S::NnSEP3 transgenic Arabidopsis had very early flowering time and produced carpeloid organ,suggesting that NnSEP3 is involved in floral development.In general,the techniques of omic and molecular biology were combined to investigate the molecular mechanism of floral organ petaloid in lotus,and explored the related genes of stamen petaloid and carpel petaloid regulation,to understand the mechanism underlying novel flower shape in lotus breeding programs.