Abstract

Floral color polymorphism can provide great insight into species evolution from a genetic and ecological standpoint. Color variations between species are often mediated by pollinators and are fixed characteristics, indicating their relevance to adaptive evolution, especially between plants within a single population or between similar species. The orchid genus Pleione has a wide variety of flower colors, from violet, rose-purple, pink, to white, but their color formation and its evolutionary mechanism are unclear. Here, we selected the P. limprichtii population in Huanglong, Sichuan Province, China, which displayed three color variations: Rose-purple, pink, and white, providing ideal material for exploring color variations with regard to species evolution. We investigated the distribution pattern of the different color morphs. The ratio of rose-purple:pink:white-flowered individuals was close to 6:3:1. We inferred that the distribution pattern may serve as a reproductive strategy to maintain the population size. Metabolome analysis was used to reveal that cyanindin derivatives and delphidin are the main color pigments involved. RNA sequencing was used to characterize anthocyanin biosynthetic pathway-related genes and reveal different color formation pathways and transcription factors in order to identify differentially-expressed genes and explore their relationship with color formation. In addition, qRT-PCR was used to validate the expression patterns of some of the genes. The results show that PlFLS serves as a crucial gene that contributes to white color formation and that PlANS and PlUFGT are related to the accumulation of anthocyanin which is responsible for color intensity, especially in pigmented flowers. Phylogenetic and co-expression analyses also identified a R2R3-MYB gene PlMYB10, which is predicted to combine with PlbHLH20 or PlbHLH26 along with PlWD40-1 to form an MBW protein complex (MYB, bHLH, and WDR) that regulates PlFLS expression and may serve as a repressor of anthocyanin accumulation-controlled color variations. Our results not only explain the molecular mechanism of color variation in P. limprichtii, but also contribute to the exploration of a flower color evolutionary model in Pleione, as well as other flowering plants.

Highlights

  • Flower color is one of the most attractive characteristics of plants in nature

  • The molecular mechanism of flower color transition has been investigated in several species, owing to the main floral pigments having been well characterized in many plants [8,9,10,11,12,13] providing sufficient information for studying floral color formation in non-model species and the opportunity to explore the relationship between phenotypic evolution and color variations

  • For the MYB genes, to determine which of the genes belonged to which R2R3-MYB subfamily, the R2R3-MYB genes from Arabidopsis thaliana were used to conduct phylogenetic analyses (Amino acid sequence obtained from Gene Bank, accession number is listed in Supplementary Table S2), and MEGA7.0 software (Institute of Molecular Evolutionary Genetics, PA, USA) [52] was used to perform sequence aligning and construct a circular phylogenetic tree according to neighbor joining method with 1000 interactions

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Summary

Introduction

Flower color is one of the most attractive characteristics of plants in nature. With such massive variation, flower color is regarded as an evolutionarily labile trait and has been shown to contribute to plant evolution [1,2,3]. The cis-regulation of transcription factors is a crucial element to promote color divergence This result is similar with the white color formation in Primula vulgaris which is caused by different genes expression pattern rather than loss- of-function mutations leading to the lack of anthocyanin [36]. It has been suggested that the competition between the anthocyanin synthesis pathway and the flavone and flavonols pathways mainly results in substrate competition between FLS with DFR, while the FLS enzyme strengthens the metabolic flux toward the flavonols and limits anthocyanin accumulation [38] This situation has been reported in other species, such as in Paeonia ostii, a higher expression of PoFLS4 in the nearly white flowers promotes dihydroflavonols transition into flavonols [11]. FFlloowweerr ppoollyymmoorrpphhiissmm ooff PPlleeiioonnee lliimmpprriicchhttiiii iinn HHuuaanngglloonngg ppooppuullaattiioonn.. ((aa)) rroossee--ppuurrppllee flfloowweerr;; ((bb)) ppiinnkk flfloowweerr;; ((cc)) wwhhiittee flfloowweerr;; ((dd))oonneeoofftthheeppoollyymmoorrpphhiiccppooppuullaattiioonnssoonnaarroocckk

Quantitative Statistics and Flower Colorimeter Analysis
Measurement of Flower Anthocyanin
Library Preparation and Sequencing
De Novo Transcriptome Assembly Annotation
Expression Profile and RT-qPCR
Genes Related to the ABP and Phylogenetic Analyses
Structure Genes and Tfs Co-Expression Network
Findings
Conclusions

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