Identification of flavonoids and expression of flavonoid biosynthetic genes in two coloured tree peony flowers

https://doi.org/10.1016/j.bbrc.2015.02.126Get rights and content

Highlights

  • Two anthoxanthin compositions and one main anthocyanin composition were found.

  • Red petals contained higher flavonoid contents identified in this study.

  • Nine structural genes in flavonoid biosynthetic pathway were isolated.

  • The shift from white to red was resulted from downstream genes expression.

Abstract

Tree peony (Paeonia suffruticosa Andr.) has been named the “king of flowers” because of its elegant and gorgeous flower colour. Among these colours, the molecular mechanisms of white formation and how white turned to red in P. suffruticosa is little known. In this study, flower colour variables, flavonoid accumulation and expression of flavonoid biosynthetic genes of white (‘Xueta’) and red (‘Caihui’) P. suffruticosa were investigated. The results showed that the flower colours of both cultivars were gradually deepened with the development of flowers. Moreover, two anthoxanthin compositions apigenin 7-O-glucoside together with apigenin deoxyheso-hexoside were identified in ‘Xueta’ and ‘Caihui’, but one main anthocyanin composition peonidin 3,5-di-O-glucoside (Pn3G5G) was only found in ‘Caihui’. Total contents of anthocyanins in ‘Caihui’ was increased during flower development, and the same trend was presented in anthoxanthins and flavonoids of these two cultivars, but the contents of these two category flavonoid in ‘Caihui’ were always higher than those in ‘Xueta’. Furthermore, nine structural genes in flavonoid biosynthetic pathway were isolated including the full-length cDNAs of phenylalanine ammonialyase gene (PAL), chalcone synthase gene (CHS) and chalcone isomerase gene (CHI), together with the partial-length cDNAs of flavanone 3-hydroxylase gene (F3H), flavonoid 3'-hydroxylase gene (F3′H), dihydroflavonol 4-reductase gene (DFR), anthocyanidin synthase gene (ANS), UDP-glucose: flavonoid 3-O-glucosyltransferase gene (UF3GT) and UDP-glucose: flavonoid 5-O-glucosyltransferase gene (UF5GT), and PAL, UF3GT and UF5GT were reported in P. suffruticosa for the first time. Their expression patterns showed that transcription levels of downstream genes in ‘Caihui’ were basically higher than those in ‘Xueta’, especially PsDFR and PsANS, suggesting that these two genes may play a key role in the anthocyanin biosynthesis which resulted in the shift from white to red in flowers. These results would provide a better understanding of the underlying molecular mechanisms of flower pigmentation in P. suffruticosa.

Introduction

Tree peony (Paeonia suffruticosa Andr.) is a rare ornamental plant native to China with more than 1600 years history of cultivation until now. As an outstanding representative of Chinese traditionally famous flowers, P. suffruticosa is elegant and its flower colour is gorgeous, which is very popular both at home and abroad [1]. Moreover, as the main ornamental characteristics of P. suffruticosa, the flower colour is rich in diversity which can be divided into nine categories including white, pink, red, purple, black, blue, green, yellow and double colour. Among these colours, white is crystal clear, noble and elegant which can particularly win the hearts of girls, and it is also a excellent material for transgenic breeding of P. suffruticosa flower with other colour varities. In addition, red is festive and auspicious and its varieties are in the majority [2]. Until now, lots of studies have been thoroughly performed concentrating on P. suffruticosa germplasm resources [3], system evolution [4], [5], plant disease [6], [7] and cultivation physiology [8], [9]. And some researches also have been conducted on the qualitative and quantitative analysis of pigment in P. suffruticosa white petals [10], [11], [12], but the underlying molecular mechanisms especially how white turns to festive red have not been fully elucidated yet till now.

For ornamental plants, flower colour is an important quality determinant which not only affects the ornamental merit but also directly influences their commercial values. In P. suffruticosa, the chemical constituents of petal colour had been studied systematically, and the colour was determined by flavonoids including anthocyanins and multiform glycosides of flavones and flavonols [2], [10], [11], [12], [13]. Wang et al. [11] analysed anthocyanins of white Zhongyuan of P. suffruticosa cultivars, six anthocyanins including peonidin 3-O-glucoside (Pn3G), peonidin 3,5-di-O-glucoside (Pn3G5G), cyanidin 3-O-glucoside (Cy3G), cyanidin 3,5-di-O-glucoside (Cy3G5G), pelargonidin 3-O-glucoside (Pg3G) and pelargonidin 3,5-di-O-glucoside (Pg3G5G), and six anthoxanthin glycosides were identified. Whereas Fan et al. [12] found only few anthocyanins, and their composition could not be characterized. Also flavonoids in red series P. suffruticosa cultivars were analyzed in more detail. All these informations provided a physiological and biochemical basis to investigate the molecular mechanisms of white formation and how white turns to red in the flower petal of P. suffruticosa.

Flavonoids formation and accumulation originated from flavonoid biosynthetic pathway has been well characterized, and extensive studies have also been carried on related enzymes and genes [14]. Up till now, this metabolic pathway has been well studied in Helianthus annuus [15], Chrysanthemum grandiflorum [16], Paeonia lactiflora [17] and other ornamental plants. In P. suffruticosa, Zhou et al. [18], [19], [20], [21] firstly isolated PsCHS, PsCHI and PsDFR, and investigated their functions. Subsequently, the full-lengths of six structural genes including PsCHS, PsCHI, PsF3H, PsF3′H and PsDFR were cloned and the putative flavonoid biosynthetic pathway was predicted and summarize by Zhang et al. [22], which were used to reveal the molecular mechanism of colour fading of in-vase P. suffruticosa ‘Luoyang Hong’ flowers comparing with on-tree ones in field conditions. This report clarified the molecular mechanism of red degree transfromation from dark to light, but not shift from red to white in P. suffruticosa. Unlike the model plant Arabidopisis thaliana, it was very difficult to find a flower colour mutation in P. suffruticosa. Therefore, in order to clarifiy the molecular mechanisms of white formation and how white turns to red in P. suffruticosa, two representative cultivars ‘Xueta’ (white) and ‘Caihui’ (red) were selected to measure flower colour variables, determine flavonoid accumulation with high-performance liquid chromatograph-electrospray ionization-mass spectrometry (HPLC-ESI-MSn), isolate nine structural genes involved in flavonoid biosynthetic pathway by rapid amplification of cDNA ends (RACE) and reverse-transcription polymerase chain reaction (RT-PCR) technologies, and investigate their expression patterns using real-time quantitative polymerase chain reaction (Q-PCR). These findings could enrich the understanding of flower pigmentation in P. suffruticosa.

Section snippets

Plant materials

Tree peony was grown in the germplasm repository of Horticulture and Plant Protection College, Yangzhou University, Jiangsu Province, China (32°30′ N, 119°25′ E). Two cultivars based on their flower colour phenotypes, including a white cultivar ‘Xueta’ and a red one ‘Caihui’ were used as the plant materials. The young leaves were used for gene isolation and the petals were used for flavonoid and gene expression analysis. After measurement of flower quality and colour indices, all samples were

Flower quality and colour indices

Two cultivars of P. suffruticosa with two different flower colours (white, ‘Xueta’; red, ‘Caihui’) were selected as the materials, and their flower diameter and flower fresh weight between ‘Xueta’ and ‘Caihui’ were shown in Fig. 1. During flower development, these two indices both gradually increased and reached maximum values in S4. and ‘Caihui’ was always larger than that of ‘Xueta’ in each stage. Moreover, their colour variables were also measured, which were expressed as . The value of

Discussion

Flower is an important organs in ornamental plants, its quality changes with certain regularity during its development. When colour variable was concerned, the petal colour of red cotton flower deepened dramatically after blooming [30], and in P. lactiflora, its petal colour in different blooming stages was measured by the Royal Horticultural Society Colour Chart (R.H.S.C.C.), and its corresponding colour was changed from red-purple to violet [31], which had been proved by Zhao et al. [17]. In

Conflict of interest

None.

Acknowledgments

This work was supported by the Agricultural Science & Technology Independent Innovation Fund of Jiangsu Province (CX[14]2135) and the Priority Academic Program Development from Jiangsu Government.

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