| Lotus, belonging to Nelumbonaceae and Nelumbo, is a kind of old and aquatic perennial herbaceous flowers. Flower colour is one of the major ornamental elements and determines the ornamental value of lotus. Nelumbo contains two species:lotus (N. nucifera Gaertn) and American lotus (N. lutea Pers). American lotus only have yellow flower, while lotus, also named as Chinese lotus, contain red, pink, white and red/white pied colours. To date, there are few studies focusing on lotus flower colouration. In this study, we conducted systematic studies to assess the contribution of flavonoid pigments to lotus petals colour and uncover the coloration mechanism of lotus petals. Firstly, the contents and constituents of anthocyanins, flavones and flavonols were analysed through high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) in 108 lotus cultivars with five kinds of colour. Then two lotus cultivar ’Yehonglian’ with red petal and ’Baige’ with white petal, were selected as materials to show the mechanisms underlying the differential accumulation of flavonoid pigments. The results are as follows:(1) Totally, five anthocyanins and fourteen flavones and flavonols were detected in different lotus germplasm. The anthocyanins were delphinidin 3-O-glucoside (Dp-3-glu), cyanidin 3-O-glucoside (Cy-3-glu), petunidin 3-O-glucoside (Pt-3-glu), peonidin 3-O-glucoside (Pn-3-glu) and Mv-3-glu (malvidin 3-O-glucoside). And flavones and flavonols were myricetin 3-O-galatoside (Myr-3-Gal), myricetin 3-O-glucoside (Myr-3-Glu), quercetin 3-Oarabinopyanosyl-(1â†'2)-galactop-yranoside (Qc-3-Ara-Gal), myricetin3-O-glucuronide(Myr-3-Gln),quercetin-O-rhamopyranosyl-((1â†'6)-galactopy-ranoside (rutin), quercetin 3-O-galacoside (hyperoside), quercetin 3-O-glucoside (isoquercitrin), quercetin 3-O-glucuronide (Qc-3-Gln), isorhamnetin 3-O-rutinoside (Iso-3-Rut), kaempferol 3-O-galactoside (Kae-3-Gal), kaempferol 3-O-glucoside (astragalin), isorhamnetin 3-O-glucoside (Iso-3-Glu), kaempferol 3-O-glucuronide (Kae-3-Gln) and isorhamnetin 3-O-glucuronide (Iso-3-Gln). Based on the aglycones, the fourteen flavones and flavones can be classified into four groups:myricetin derivatives (Myr-D), Quercetin derivatives (Qc-D), kaempferol derivatives (Kae-D) and isorhamnetin derivatives (Iso-D).(2) In general, the yellow and white cultivars hardly contained any anthocyanins, while two or three kinds of anthocyanins can be detected in several red/white pied or yellow cultivars. Five anthocyanins could be detected in almost red and pink cultivars, with the former containing more than the latter. Among the five anthocyanins, the most abundant one was Dp-3-glu, followed by Mv-3-glu. Except for the yellow cultivars, Kae-D was the most abundant one among flavones and flavonols. While Iso-D were the most abundant in yellow cultivars.(3) Selecting two lotus cultivars—’Yehonglian’(red flower) and’Baige’(white flower) as representative cultivars, Their petals were firstly analysed by HPLC-MS, and five anthocyanins, fourteen flavones and flavonols were identified in’Yehonglian’petals, but no anthocyanin and 13 flavones and flavonols were detected in’Baige’petals. Both ’Yehonglian’and’Baige’petals contained proanthocyanidins (PA). Then, the pigments accumulations in the transections of’Yehonglian’and’Baige’were observed through light microscope observation, and red pigment accumulation were observed in the upper epidermis cells of’Yehonglian’petals, while no pigment accumulation was detected in ’Baige’petals.(4) Then, the comparative protemic analyses of petals between red’Yehonglian’and white’Baige’were performed, and a total of 88 differentially expressed proteins were identified by MAIDI-TOF-MS/MS, with 36 more abundant and 52 less abundant in ’Yehonglian’than’Baige’. Among the all identified proteins, four were the key enzymes that invove in anthocyanin pathway, including flavanone 3-hydroxylase (F3H), anthocyanidin synthase (ANS), anthocyanidin 3-O-glucosyltransferase (UFGT) and glutathione S-transferase (GST). F3H and UFGT were more abundant in’Baige’, while ANS and GST were more abundant in’Yehonglian’.(5) The transcriptional level of structural genes in anthocyanin pathway were analysed, and showed that chalcone synthase (CHS), flavanone 3’-hydroxylase (F3’H), F3H and UFGT expressed higher in’Baige’; while chalcone isomerase (CHI), flavanone 3’5’-hydroxylase (F3’5’H), dihydroflavonol reductase (DFR), ANS and GST expressed higher in’Yehonglian’. What’s more, the transcriptional level of F3H and UFGT were consistant with the protein expression level which were higher in’Baige’, and ANS and GST also had the same expresion profile at both transcriptional level and protein level with higher expression in’Yehonglian’.(6) The sequences of the open reading frame (ORF) and 2000 bp promoter of ANS gene were analysed in the petals of’Yehonglian’ and’Baige’, the results showed that there were no differences between these two cultivars. But the methylation sites and levels of ANS promoter existed differences between’Yehonglian’ and ’Baige’. And ’Baige’ had 18 methylation sites (including 14 CQ 2 CHH and 2 CHG) more than ’Yehonglian’ and its total methylation level was higher than that of ’Yehonglian’.Based on these results, we can draw conclusions as follows:(1) The red colour of lotus petals was determined by the accumulation of anthocyanin. The higher content of total anthocyanins petals contains, the darker in red colour it is. Iso-D seemed to contribute a litter to the yellow colour of lotus petals, which is also the difference in the flavonoids distribution between Asial lotus and American lotus.(2) The differences of lotus petal colouration between red’Yehonglian’ and white ’Baige’ might be caused by the different methylation level of the promoter region of ANS gene, which might influence the binding between promoter region and transcription factors.This study not only assessed the flavonoid pigment in the petals of lotus germplasm, but also studied on the mechanism of lotus petal colouration. The results might provide some new insights into the mechnism of flower pigmentation in lotus, and be helpful for lotus breeding. |
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