Identification of differentially expressed genes implicated in peel color (red and green) of Dimocarpus confinis

The tropical/subtropical fruit tree longan (Dimocarpus longan Lour.) is in the family Sapindaceae that is cultivated all over the world, especially in China, Thailand, Vietnam (Jiang et al. 2002). Nowadays, with the rapid development of the agricultural economy, planting area and field in China has been the largest and highest in the world so far (Wu 2010).

Peel, the pulp section, differentiated and developed from ovary wall. Mature pericarp is generally divided into exocarp, mesocarp, endocarp. As reported, most peels are likely to have a certain characteristics, such as medical use, value-added ingredients for various food applications, anti-mosquito and deodorant (Abdul Aziz et al. 2012; Denis et al. 2013; Rawson et al. 2014). Additionally, another trait of peel is about color, which is one of the main factors that determines consumer preference and market price. Peel color of many other fruits except longan may be varied with the environmental or internal factors changed (González-Talice et al. 2013; Liu et al. 2015; Zhao et al. 2011). In the aspect of external factors, peach peel color changed when processed by bagging with a widely applied Yellow-Paper (Liu et al. 2015), and the intensity of light also plays an fundamental role in color development of apple peel (Zhao et al. 2011). As for the internal factors, pigments, total phenolic and total flavonoid concentration are important factors determining color and internal apple quality (González-Talice et al. 2013), and more and more researches showed anthocyanins (ACs) played an important role in peel color (Liu et al. 2015; Rahim et al. 2014; Wang et al. 2015; Zhao et al. 2013). ACs, a naturally water-soluble pigment of flavonoid family generated from secondary metabolites, are widely distributed in fruits and vegetables, as well, its potential health benefits to humankind provoking an increasing interest in these compounds (Boyer and Hai Liu 2004; Hyson 2011; Stover and Mercure 2007). Anthocyanin plays a photoprotective role in plants under high light or photoinhibition conditions (Close and Beadle 2005; Hoch et al. 2003; Hughes et al. 2005, 2007, 2012; Li et al. 2008; Manetas et al. 2002; Williams et al. 2003). In pear, the higher photoprotective capacity in the sun-exposed peel of red “Anjou” pear than green “Anjou” is mainly attributed to its higher anthocyanin concentration (Li et al. 2008). But, do anthocyanin act on red peel (RP) and green peel (GP) of longan, except for photoprotective of some other fruits? And which of genes involved in anthocyanin biosynthetic pathways play a major role in peel coloration? The structural genes, encoding corresponding enzymes in the anthocyanin biosynthetic pathway, have been cloned from varieties of plants, and several regulatory genes implicated in the activation of coloration have recently been cloned in previous studies as well (Espley et al. 2007; Goff et al. 1992; Goodrich et al. 1992; Niu et al. 2010; Schwinn et al. 2006). In apple, there were two cultivars with red and green peel, anthocyanins and flavonols elevated when turning shaded peel (shaded peel of the two cultivars were green) to sun exposure for a week, along with green peel to red peel. As well, exposure of the shaded peel to full sun caused marked up-regulation of expression levels of MYB10 (a transcriptional factor in the regulation of anthocyanin biosynthesis) and seven structural genes in anthocyanin synthesis (PAL, CHS, CHI, F3H, DFR1, LDOX, and UFGT) (Feng et al. 2013). Besides, myeloblastosis (MYB) was also proved to play an important role in regulating peel color in some fruits, such as peach, pear, apple (Feng et al. 2013; Rahim et al. 2014; Sun et al. 2013; Yang et al. 2015), meanwhile, MYB10.1 and MYB10.3 have positive correlation with the expression of key structural genes of the anthocyanin pathway in peach, such as chalcone synthase flavanone 3-hydroxylase (F3H), and UDP-glycose: flavonoid glycosyltransferase (UFGT) (Rahim et al. 2014). PyMADS18 was reputed to be involved in anthocyanin accumulation and regulation of anthocyanin synthesis in early fruit development of pear (Wu et al. 2013), and anthocyanidin synthase (ANS) and UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT), whose different expressions led to the coloration differences between occidental and oriental pears, were speculated to be key genes for anthocyanin biosynthesis for red-skinned pear (Yang et al. 2015).

In our previous study, anthocyanin content and composition in the peel of Dimocarpus confinis (Jiang et al. 2014), a relative species of Dimocarpus Lour., were examined using a HPLC method. The results showed that anthocyanin content was 18.60 ± 5.12 mg kg⁻¹ (FW) in red peel, and was significantly higher than in light red peel and in blue green peel by 6.8 times and 33.2 times, respectively. In the present study, a special longan germplasm resource of Longli from Fujian Province, whose fruitlet peels showed red and green in the fruit development process individually (Fig. 1a, b), were applied to initially revealing the molecular mechanism of regulating peel color. So in this study, we firstly sequenced the transcriptomes of Red Peel and Green Peel longan using Illumina technology. We focused on the discovery of encoding enzymes involved in the anthocyanin biosynthetic pathway and obtained sets of up-regulated and down-regulated genes from red and green peel of longan, and finally identified some candidate genes related to anthocyanin synthesis in longan peel. The assembled annotated transcriptome sequences provide a valuable genomic resource to further understand the molecular mechanism of regulating peel color.

Coloration of exocarp is mainly associated with accumulation type, quantity and distribution of anthocyanin, whose synthesis mechanism is not only about basic research of the fruit industry, it is also about consumer orientation. So far, there is no completely parallel key factor acting on anthocyanin biosynthesis pathway among different species, even the same. All the different/same species have their own specific expressive properties, especially the fruit types of aril, such as longan, litchi, representing significant differences of anthocyanins biosynthesis pathway from other fruits. Therefore, making them clear whether structural genes express in peel of special longan germplasm resource, which genes are the key ones for coloration of longan peel, and how the synthetic genes co-expressed is of great significance for elucidating the molecular basis of coloration of longan peel.

Over the past decade, the development of transcriptomic and genomic technologies has contributed to a better understanding of coloration of fruit peel at the molecular level. However, most of our knowledge about peel coloration has arisen from studying coloration regulatory genes in many other fruits except longan. A lack of genetic information hinders our research, but at the same time, it also makes our study more essential.

GO annotation, whose functional interpretations for plants are primarily based on the Arabidopsis thaliana genome, is able to provide a description of gene products in terms of their associated molecular functions, cellular components, and biological processes (Berardini et al. 2004). The GO annotations of the unique genes were most frequently related to biological processes (29,618 unigenes), followed by molecular function (12,489 unigenes) and cellular components (8036 unigenes). For each unigene, the specifically annotated GO terms provide a broad overview of the groups of genes cataloged in the transcriptome, and the GO annotations provided valuable clues to investigate the biological processes, molecular functions, and cellular structures of the Dimocarpus Lour. transcriptome.

The KEGG classification system, integrating current knowledge on molecular interaction networks, provides an alternative functional annotation of genes according to their associated biochemical pathways (Kanehisa et al. 2004). Metabolic pathways were well represented among longan peel unique sequences, most of which were associated with carbohydrate metabolism, translation, folding, sorting and degradation. Overview, amino acid metabolism, environmental adaptation, lipid metabolism, energy metabolism and biosynthesis of secondary metabolites were included in the top 19 pathways. Flavonoid biosynthesis is an integral part of secondary metabolism, and the transcripts encoding some enzymes involved in the flavonoid biosynthesis pathway, such as DFR, F3H, and ANS were present in our Illumina sequences dataset (Table 2); therefore, flavonoid biosynthesis pathway should be considered within the context of peel coloration of longan.

With the aim of identifying genes involved in regulating peel color, RNA sequencing was used to examine differentially expressed genes between “RP” and “GP” longan in this study, and we speculated the peel color of longan may be involved in the flavonoid metabolism pathway, especially the anthocyanin biosynthesis.

DFR, F3H and ANS are parts of important steps in the flavonoid biosynthetic pathway of anthocyanins, respectively, they convert dihydroflavonol into leucoanthocyanidin, naringenin into dihydrokaempferol, leucoanthocyanidin into anthocyanin in the anthocyanin biosynthetic pathway (Ahmed et al. 2014; Holton and Cornish 1995; Lin et al. 2013). Especially, DFR, whose expression abundances reached to 16-fold change in red peel compared with green peel, presented the most significantly different expression among DFR, F3H and ANS. DFRA is the key enzyme for the biosynthesis of anthocyanins in the skins of peach and nectarine fruit (Zhou 2009). In addition, regulating the expression of DFR can change the flower color in Japanese parsley, tobacco and petunia (Yamaguchi et al. 2004). The substrate specificity of the DFR often determines which anthocyanidins a plant accumulates (Magnus 1999). In purple kale (Brassica oleracea var. acephala f. tricolor), the expression of anthocyanin biosynthetic gene DFR is enhanced in response to low temperature treatment (Zhang et al. 2012). In conclusion, the DFR gene has different expression pathways in different plant species; therefore, the function of DFR in the anthocyanin biosynthesis process remains to be further investigated.

The CYP75A1 gene, belongs to the CYP75A subfamily, which is able to catalyze the 3′5′-hydroxylation of naringenin and eriodictyol to form 5,7,3′,4′,5′-pentahydroxy flavanone and 3′,5′-hydroxylation of dihydrokaempferol and dihydroquercetin to form dihydromyricetin. Flavonoid 3′,5′-hydroxylase( F3′5′H) is necessary for biosynthesis of the anthocyanins that confer a violet or blue color to most plants (Tanaka et al. 2008). In berry, F3′5′H gene expression has a functional impact on anthocyanin biosynthesis that persists during fruit ripening, and among red grape varieties, expansion and sub-functionalization of F3′5′Hs have increased the complexity and diversification of the fruit color phenotype (Falginella et al. 2010). As well, in the grapevine lineage, higher levels of F3′5′Hs transcription in dark blue cultivars than light red cultivars, even in green-peel cultivars, F3′5′H transcripts are completely absent (Mattivi et al. 2006; Pomar et al. 2005). In this study, expression abundance of CYP75A1 was very low in RP longan, further study should focus on its biologically significance combined with related background knowledge.

C1 is a regulatory gene of the anthocyanin pathway, which regulates the expression of at least three structural genes: chalcone synthase, dihydroflavonol reductase and flavonol O₃ glucosyltransferase. In the past decades, research on C1 gene mainly focus on maize (Zea mays) (Avila et al. 1993; Franken et al. 1994; Köhler et al. 1995; Petroni et al. 2000; Piazza et al. 2002; Scheffler et al. 1994). In maize, C1 is required for anthocyanin synthesis only in seed tissues, and different light treatments affected the expression level, white, red, and blue light were effective in stimulating anthocyanin accumulation and expression of the MYB-related gene (Piazza et al. 2002). The accumulation of C1 transcript is under both developmental and light control (Franken et al. 1994), and in recent years, many studies indicated that MYB played an important role in regulating peel color (Espley et al. 2007; Feng et al. 2013; Niu et al. 2010; Rahim et al. 2014; Schwinn et al. 2006; Sun et al. 2013; Yang et al. 2015). In our study, C1 gene was detected as a significantly DEG, we presumed the light regulation of transcription factors may control anthocyanin biosynthesis in longan peel.

We have been aware that genetic evidences are needed to support these hypothesis on function gene DFR, F3H, ANS, CYP75A1 and C1, which were speculated to play an important role in formation of peel color in longan. The expression abundance of DFR, F3H, ANS, CYP75A, and C1 and the accumulation of anthocyanin in RP and GP are worthy of further investigation, as well as their SNPs. With the aim of revealing the molecular mechanism of regulating peel color deeply in woody plants, the quantitative real-time PCR (qPCR) and high-performance liquid chromatography (HPLC) analysis should be respectively applied to verifying the expression levels of these genes and total anthocyanin concentration (Additional file 1).

To summarize, this work is the first report of gene-expression profiling in longan skin conducted by Illumina next-generation sequencing technology. We identified the genes encoding key enzymes involved in flavonoid biosynthesis pathways, which are most likely to play an important role in peel color of longan. Besides, the accumulation of flavonoids and the expression levels of genes associated with their biosynthesis and metabolism in longan peel are worthy of further investigation, which could help provide insights into the molecular mechanisms of regulating peel color in woody plants (Additional file 2).