Spatiotemporal Transcriptome Analysis Provides Insights into Bicolor Tepal Development in Lilium “Ti

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    近期,中国农业科学院蔬菜花卉研究所百合课题组继揭示百合紫色子房形成的分子机理之后又揭示了双色百合花形成的关键机理。相关研究成果近期在线发表于国际知名期刊《植物科学前沿(Frontiers in Plant Science)》上。
    双色百合花是指在同一片花被片上显现两种不同的花色,与单色花相比具有更丰富的花色及其奇特的色彩变化,观赏价值更高,有可能在吸引昆虫方面也更具效率。百合品种小飞碟 (Tiny Padhye) 双色花被片发育的过程中,花被片下部逐渐由绿色变为紫红色,而上部由绿色变为白色。这类百合双色花形成的分子机理在花色研究领域鲜见报道。而双色花形成的分子机理的揭示可以为花色的人工调控及分子改良提供理论依据。



Spatiotemporal Transcriptome Analysis Provides Insights into Bicolor Tepal Development in Lilium “Tiny Padhye”

Leifeng Xu1, Panpan Yang1,2, Yayan Feng1, Hua Xu1, Yuwei Cao1, Yuchao Tang1, Suxia Yuan1, Xinyan Liu1 and Jun Ming1*

1Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
2Department of Ornamental Plants, College of Landscape Architecture, Nanjing Forestry University, Nanjing, China

The bicolor Asiatic hybrid lily cultivar “Tiny Padhye” is an attractive variety because of its unique color pattern. During its bicolor tepal development, the upper tepals undergo a rapid color change from green to white, while the tepal bases change from green to purple. However, the molecular mechanisms underlying these changes remain largely uncharacterized. To systematically investigate the dynamics of the lily bicolor tepal transcriptome during development, we generated 15 RNA-seq libraries from the upper tepals (S2-U) and basal tepals (S1-D, S2-D, S3-D, and S4-D) of Lilium “Tiny Padhye.” Utilizing the Illumina platform, a total of 295,787 unigenes were obtained from 713.12 million high-quality paired-end reads. A total of 16,182 unigenes were identified as differentially expressed genes during tepal development. Using Kyoto Encyclopedia of Genes and Genomes pathway analysis, candidate genes involved in the anthocyanin biosynthetic pathway (61 unigenes), and chlorophyll metabolic pathway (106 unigenes) were identified. Further analyses showed that most anthocyanin biosynthesis genes were transcribed coordinately in the tepal bases, but not in the upper tepals, suggesting that the bicolor trait of “Tiny Padhye” tepals is caused by the transcriptional regulation of anthocyanin biosynthetic genes. Meanwhile, the high expression level of chlorophyll degradation genes and low expression level of chlorophyll biosynthetic genes resulted in the absence of chlorophylls from “Tiny Padhye” tepals after flowering. Transcription factors putatively involved in the anthocyanin biosynthetic pathway and chlorophyll metabolism in lilies were identified using a weighted gene co-expression network analysis and their possible roles in lily bicolor tepal development were discussed. In conclusion, these extensive transcriptome data provide a platform for elucidating the molecular mechanisms of bicolor tepals in lilies and provide a basis for similar research in other closely related species.

Lily (Lilium spp.) is one of the most important ornamental plants because of their large flowers with unique and diverse colors. Hybrid lily cultivars are grouped according to their genetic phylogeny (Shahin et al., 2014). Asiatic hybrids, one of the major groups of hybrids, are derived from interspecific crosses among species in the section Sinomartagon that are mainly distributed in China. A typical ornamental feature of Asiatic hybrid lilies is the variety of flower colors, including yellows, oranges, pinks, reds, and whites. In addition to the various colors, Asiatic hybrid lilies also exhibit variations in pigmentation patterns, including spots and bicolors.

Bicolor flowers have fascinating color patterns. Some of the molecular mechanisms leading to the development of bicolor petals have been identified. Post-transcriptional gene silencing (PTGS) of chalcone synthase (CHS) genes in non-pigmented areas produces the white areas of bicolor flower petals in several horticultural crops, such as petunia (Petunia hybrida) (Koseki et al., 2005; Saito et al., 2006; Morita et al., 2012), camellia (Camellia japonica) (Tateishi et al., 2010), and dahlia (Dahlia variabilis) (Ohno et al., 2011). Two types of bicolor flowers are found in Asiatic hybrid lilies; in one type, anthocyanins accumulate in the upper tepals (e.g., “Lollipop”) while in the other type, anthocyanin pigments accumulate in the tepal bases (e.g., “Tiny Padhye”). Recently, Suzuki et al. (2016) showed that the first type of bicolor tepals (“Lollipop”) resuled from the transcriptional regulation of anthocyanin biosynthetic genes. However, whether the same molecular mechanisms underlie the second type of bicolor tepals remains unclear.

Anthocyanins, a class of flavonoid compounds, are responsible for the pink, red, blue, and purple colors in diverse plant tissues (flowers, fruits, leaves, etc.) (Davies et al., 2012; Zhao and Tao, 2015). The anthocyanin biosynthetic pathway is one of the best-known specialized metabolic pathways and most of the anthocyanin biosynthetic genes have been characterized in different plant species (Winkel-Shirley, 2001; Grotewold, 2006). There have been several reports on the molecular mechanisms of the anthocyanin biosynthetic pathway in lilies. Three CHS genes (LhCHSA, LhCHSB, and LhCHSC), LhDFR, LhPAL, LhF3H, LhF3′H, LhANS, and two R2R3-MYB transcription factors (TFs; LhMYB6 and LhMYB12) were isolated from the tepals of Asiatic lily “Montreux” (Nakatsuka et al., 2003; Yamagishi et al., 2010; Lai et al., 2012). LhMYB12-Lat and LrMYB15 were shown to determine the unique anthocyanin color patterns of the Tango Series cultivars of Asiatic hybrid lilies (Yamagishi et al., 2014) and Lilium regale (Yamagishi, 2016), respectively. However, there is still limited information on the overall molecular mechanisms underlying tepal pigmentation in lilies.

The petals of some flowering plants contain chlorophyll (Chl) in the early developmental stages. As the petals mature, the Chl content gradually decreases during the late developmental stages (Ohmiya et al., 2014). The Chl metabolic pathway is relatively well-characterized in many plant species. This pathway has three phases: Chl a synthesis, interconversion of Chl a and Chl b (Chl cycle), and Chl degradation (Eckhardt et al., 2004; H?rtensteiner, 2013). The molecular mechanisms of Chl metabolism in leaves and fruit have been largely unraveled (Lim, 2003; Lai et al., 2015; Wen et al., 2015). Ohmiya et al. (2014) showed that low rates of Chl biosynthesis and high rates of Chl degradation led to the absence of Chls in non-green carnation petals. However, the molecular mechanisms regulating Chl metabolism in lily petals are still unknown.

Whole-transcriptome sequencing based on next-generation sequencing has become a powerful tool to identify candidate genes and investigate gene expression patterns of non-model organisms without reference sequences (Mutz et al., 2013). Furthermore, transcriptome data can be used to identify candidate genes relevant to a given pathway or phenotype using a weighted gene co-expression network analysis (WGCNA). WGCNA is a powerful approach for finding clusters (modules) of highly correlated genes with a high topological overlap (Langfelder and Horvath, 2008; Zhao et al., 2015). This strategy has been used to identify regulators and co-expression networks in Arabidopsis thaliana (Appel et al., 2014), strawberry (Fragaria spp.) (Hollender et al., 2014), apple (Malus × domestica) (Bai et al., 2015), and sweet orange (Citrus sinensis L. Osbeck) (Huang et al., 2016).

In this study, RNA samples from the upper tepals (stage 2; S2) and tepal bases (S1–4) of “Tiny Padhye” bicolor tepals were sequenced using the Illumina sequencing platform. Global gene expression profiles, focusing mainly on genes in the anthocyanin biosynthetic pathway and Chl metabolic pathway, during bicolor tepal development were analyzed using a differential gene expression strategy and quantitative real-time PCR (qRT-PCR) analyses. We aimed to identify structural genes and TFs associated with the anthocyanin biosynthetic pathway and Chl metabolic pathway, and to investigate their spatiotemporal expression patterns during bicolor tepal development to characterize the mechanisms of bicolor (white and purple) tepals development in lilies.