Overexpression of LiTPS2 from a cultivar of lily (Lilium ‘Siberia’) enhances the monoterpenoids content in tobacco flowers

Highlights

• Three unigenes related to TPS genes were selected through transcriptomes of petals at different flowering stages and leaves.

• Lilium ‘Siberia’ main monoterpenes components are synthesized by LiTPS2 synthases.

• LiTPS2 could utilize geranyl pyrophosphate to produce linalool and other monoterpenes, and farnesyl pyrophosphate to form trans-nerolidol and other sesquiterpenes, respectively.

• Ectopic expression in transgenic tobacco plants suggested that LiTPS2 contributed the synthesis of monoterpenes, such as, linalool, myrcene and (E)-β-ocimene in flowers of transgenic tobacco.

Abstract

Lily, a famous cut flower with highly fragrance, has high ornamental and economic values. Monoterpenes are the main components contributing to its fragrance, and terpene synthase (TPS) genes play critical roles in the biosynthesis of monoterpenoids. To understand the function of TPS and to explore the molecular mechanism of floral scent in cultivar Lilium ‘Siberia’, transcriptomes of petal at different flowering stages and leaf were obtained by RNA sequencing and three unigenes related to TPS genes were selected for further validation. Quantitative real-time PCR showed that the expression level of LiTPS2 was greater than that of the other two TPS genes. Phylogenetic analysis indicated that LiTPS2 belonged to the TPSb subfamily, which was responsible for monoterpenes synthesis. Subcellular localization demonstrated that LiTPS2 was located in the chloroplasts. Furthermore, functional characterization showed that LiTPS2 utilized both geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) to produce monoterpenoids such as linalool and sesquiterpenes like trans-nerolidol, respectively. Ectopic expression in transgenic tobacco plants suggested that the amount of linalool from the flowers of transgenic plants was 2–3 fold higher than that of wild-type plants. And the emissions of myrcene and (E)-β-ocimene were also accumulated from the flowers of LiTPS2 transgenic lines. Surprisingly, these three compounds were the main fragrance components of oriental lily hybrids. Our results indicated that LiTPS2 contributed to the production of monoterpenes and could effectively regulate the aroma of Lilium cultivars, laying the foundation for biotechnological modification of floral scent profiles.

Introduction

Often termed the ‘soul of flowers’, floral scent is an important quality indicator for ornamental plants. Both the floral and vegetative parts of many plant species emit volatile compounds with distinctive aroma. Floral scent is composed of a mixture of these volatile organic compounds (VOCs) (Knudsen, 2006), which can protect plants against herbivory, by acting as toxins, and provide chemical signals to pollinators or abiotic stress responses (Holopainen and Gershenzon, 2010). According to their metabolic origins, VOCs are divided into three major classes: terpenoids, phenylpropanoids/benzenoids, and fatty acid derivatives (Dudareva and Pichersky, 2000).

Terpenoids, as the largest volatile class in plants (Vranova et al., 2012), are produced from two different biosynthetic pathways in plant cells: (1) the mevalonic acid (MVA) pathway that is involved in the synthesis of sesquiterpenes and triterpenes in cytosol; and (2) the methylerythritol phosphate (MEP) pathway which is related to the biosynthesis of monoterpenes, diterpenes, and carotenoids in the plastid (Pichersky et al., 1995). These two pathways are divided into three main stages: 1) production of intermediates isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP); 2) production of the terpenoid precursors GPP, FPP and geranylgeranyl pyrophosphate (GGPP); and 3) after the production of these precursors, most of terpenoids are synthesized by TPSs (Trapp and Croteau, 2001).

TPSs are the primary enzymes in the synthesis of terpenoids, and the transcriptional activity of the corresponding TPS gene greatly influences the production of terpenes (Tholl, 2006). To date, more than 200 monoterpene and sesquiterpene synthase genes have been identified (Degenhardt et al., 2009). And various TPSs in several plants with different structures and functions, including multiple-product and single-product enzyme, have been characterized (Dudareva and Pichersky, 2000). Multiple-product enzyme can utilize different substrates including GPP, geranyl diphosphate (GDP), FPP, and farnesyl diphosphate (FDP) to form multiple terpenoids (Pazouki et al., 2015). For example, AaTPS6 from Artemisia annua catalyzed the formation of 1,8-cineole, sabinene, and other seven terpenoids (Ruan et al., 2016). CsTPS1 from Citrus sinensis was able to produce a mixture of monoterpenes (high amounts of β-myrcene and small amounts of limonene) using GPP as a substrate (Borghi and Xie, 2018). And 1,8-cineole synthase (Cin) from Nicotiana tabacum, could catalyze GPP to produce seven terpenes (Roeder et al., 2007). Single-product enzymes have also been reported, such as S-linalool synthase from Clarkia breweri catalyzing GPP to produce linalool (Lucker et al., 2001) and AaTPS2 from A. annua produced β-myrcene only (Ruan et al., 2016). Except for that, overexpressed TPSs could enhance the emission amounts of the volatile terpene. Overexpression of the geraniol synthase (GES) gene from Valeriana officinalis and LdGES from Lippia dulcis in transgenic tobacco plants resulted in an increased amount of geraniol and oxidized geraniol compounds (Dong et al., 2013). However, the contribution of TPS genes to the production of monoterpenes in lily needs further investigation.

Lily is a fragrant cut flower with high ornamental values. Many Lilium sp. Cultivars emit a large amount of volatile monoterpene compounds, which can attract the attention of consumers (Johnson et al., 2016). And monoterpene compounds, including myrcene, (E)-β-ocimene and linalool, are the major compounds of scented lilies (Kong et al., 2012). High-throughput RNA sequencing (RNA-Seq) has been used as an effective analysis tool for revealing molecular mechanism and understanding gene regulatory network in plants (Fan et al., 2015). RNA-Seq has been used not only to analyze the cold stress response in L. lancifolium (Deng et al., 2014), the carbohydrate metabolism during bulblet formation and development in L. davidii var. unicolor (Li et al., 2014), but also to understand the different fragrance mechanisms between the cultivar L. ‘Siberia’ with a strong fragrance and L. ‘Nonano’ with a faint fragrance (Hu et al., 2017). In addition, RNA-Seq, combined with two-dimensional analysis and weighted gene co-expression network analysis (WGCNA), has been used as a tool for screening candidate genes related to the formation of flower fragrance (Shi et al., 2018). Although the majority of differentially expressed genes (DEGs) have been identified, few studies focused on their functions, particularly the TPS genes downstream the MEP pathway (Trapp and Croteau, 2001), or on the molecular regulation of monoterpene biosynthesis in lilies. To overcome these knowledge gaps, the transcriptomes of petal at different flowering stages and leaf of the cultival L. ‘Siberia’ were performed in this study. By examining the DEGs related to monoterpene synthase, LiTPS2 was selected as a candidate gene. Thus, the function of LiTPS2 in regulating the synthesis of monoterpenes in L. ‘Siberia’ was investigated, aiming to provide the theoretical basis for producing new Lilium varieties or improving existing varieties with a desirable aroma for the consumer through genetic engineering.