Flower Color Diversity Revealed by Differential Expression of Flavonoid Biosynthetic Genes in Sacred Lotus

Sacred lotus (Nelumbo nucifera) is an important aquatic ornamental plant which contains several diverse flower colors, but the underlying mechanisms of its flower coloration remain unclear. In this study, seven complementary DNA (cDNA) clones of genes involved in flavonoid biosynthesis, including chalcone synthase (CHS), chalcone isomerase (CHI ), flavanone 3-hydroxylase (F3H ), flavonoid 3#-hydroxylase (F3#H ), flavonoid 3#,5#hydroxylase (F3#5#H ), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), were isolated and characterized. Moreover, expression patterns of these seven genes and pigment profiles were investigated across four N. nucifera cultivars with different flower colors: Zhongguohongbeijing [ZGH (red)], Xinghuafen [XHF (pink)], Molingqiuse [MLQS (yellow)], and Zhufengcuiying [ZFCY (white)]. Real-time quantitative polymerase chain reaction (qRT-PCR) analysis showed that during flower development, transcripts of early biosynthetic genes (NnCHS, NnCHI, and NnF3H) were abundant at the early stage; noticeably, highest expression of NnCHI in MLQS probably induced abundant anthoxanthin synthesis and displayed yellow. Expression of late biosynthetic genes, especially NnDFR and NnANS, was generally consistent with change patterns of anthocyanins in ZGH and XHF, but NnF3#H was barely detectable in the pink cultivars. Meanwhile, negligible expression of NnDFR and NnANS was detected in MLQS and ZFCY, respectively, which blocked their colored anthocyanin biosynthesis. Spatial expression analysis revealed that most flavonoid biosynthetic genes were highly expressed in floral tissues, rather than leaves. These results suggest that in N. nucifera cultivars with different flower colors, flavonoid biosynthesis is differentially regulated by the expression of these flavonoid biosynthetic genes, among which,NnCHI,NnF3#H,NnDFR, andNnANS are supposed to be critical for pigment accumulation, and therefore, affect different flower coloration. Sacred lotus is a famous perennial aquatic plant around the world, and widely used as an ornamental flower for water features in summer. It belongs to the family Nelumbonaceae, which consists of one genus, Nelumbo, with only two species, N. nucifera and Nelumbo lutea (Wang and Zhang, 2005). Although the two species differ morphologically and genetically, there is no reproductive barrier between them, and more than 800 cultivars have been created by natural and artificial hybridization (Zhang, 2011). These cultivars possess several attractive flower colors including red, pink, white, yellow, and versicolor, but compared with other ornamental plants, the lotus flower color is not rich in diversity yet. Flower color has been regarded as a primary ornamental characteristic that determines the ornamental merit and landscaping application value. Therefore, breeding much more novel flower color cultivars is one of the most important breeding objectives in N. nucifera, and understanding genetic mechanisms of its petal coloration is a key to successful breeding. Flower coloration is due to the accumulation of pigments within epidermal cells, including flavonoids, which include chalcones, anthoxanthins (flavones and flavonols), and anthocyanins (Qi, 1989). Among them, anthocyanins have been implicated as the major coloring factors in various species (Grotewold, 2006; Tanaka et al., 2005; Zhao and Tao, 2015). In N. nucifera, chemical constituents of flower color have been systematically studied. Yang et al. (2009) identified five major anthocyanins, including delphinidin 3-O-glucoside (Dp3G), cyanidin 3-O-glucoside (Cy3G), petunidin 3-O-glucoside (Pt3G), peonidin 3-O-glucoside (Pn3G), and malvidin 3-Oglucoside (Mv3G), and 10 flavonols in six cultivars of N. nucifera. But another major aglycon for anthocyanin named pelargonidin was not detected. Furthermore, Deng et al. (2013) assessed the composition and content of flavonoids across 108 cultivars of N. nucifera with red, pink, yellow, white, and pied petal colors. Five aforementioned anthocyanins except pelargonidin were found in both red and pink cultivars, with much less cyanidin derivatives (Cy3G and Pn3G) in the pink groups, whereas 14 anthoxanthins were detected and quantified in all cultivars. All the information has provided physiological and biochemical basis to study molecular mechanisms underlying flower color formation and diversity in N. nucifera. Flavonoid formation and accumulation are derived from the flavonoid biosynthetic pathway, which has been well characterized in model species Antirrhinum majus, Petunia ·hybrida, and Arabidopsis thaliana (Grotewold, 2006; Nishihara and Nakatsuka, 2011; Tanaka et al., 2008; Winkel-Shirley, 2001). The putative flavonoid biosynthetic pathway without pelargonidin-based anthocyanins in N. nucifera is illustrated in Fig. 1. In brief, the first committed step for flavonoid synthesis is the formation of chalcone catalyzed by chalcone synthase (CHS), which is required for the synthesis of all flavonoids. Chalcone Received for publication 14 June 2016. Accepted for publication 21 Sept. 2016. This work was financially supported by the Open Fund of Key Laboratory for Genetic Improvement of Ornamental Plants in Jiangxi (no. 2013-KLB-03), the China Postdoctoral Science Foundation funded project (no. 2014M561674), the National Natural Science Foundation of China (no. 31400600), and the Funding of Agricultural Science and Technology Innovation in Jiangsu [no.