Part 2 - Relationship between the Composition of Flavonoids and Flower Colors Variation in Tropical Water Lily (Nymphaea) Cultivars

Relationships between color parameters and flavonoid compositions

The flower color is associated with type, content and physicochemical property of pigment, the pH value of vacuole, the shape of epidermis cell and organization structure in petals. However the composition of pigments is the most important one. So choosing L*, a*, b*, C* and h as dependent variable, 37 indexes included 34 contents of pigment components, TA (total anthocyanins content), TF (total flavonols and chalcones content) and CI (co-pigment index = TF/TA) as independent variable. The regression equations about the relationships between color parameters and flavonoids components were established to study the interactions between pigment composition and color formation by multiple linear regression (MLR) analysis [35]. Statistical results indicated as follows (n = 35, p<0.05):

L* = 61.074+0.032Qu3Rh+0.054Km7galloylGaRh - 0.031Dp3′galloylGa (R2 = 0.531)

a* = −3.113+0.040TA+0.065Qu3acetylGa (R2 = 0.850)

b* = −20.226−0.202Qu3acetylGa+0.007Chal2′Ga+0.016Qu3acetylRh (R2 = 0.673)

C* = 10.750+0.032TA+0.167Qu3acetylGa (R2 = 0.688)

h = −1.322+0.005Dp3galloylGa−0.001Dp3′galloylGa (R2 = 0.953)

From this MLR analysis, it can be seen that there are many factors affecting the color: TA; Dp3galloylGa; Dp3′galloylGa; Qu3Rh; Qu3acetylGa; Qu3acetylRh; Km7galloylGaRh and Chal2′Ga. TA and Qu3acetylGa had positive effects on the value of a* and C*, whereas Qu3acetylGa and Qu3acetylRh had negative effects on the value of b*, meanwhile Chal2′Ga had positive effects on the value of b*. Km7galloylGaRh and Qu3Rh had positive effects on the value of L*, but Dp3′galloylGa had negative effects on the value of L* and h. In addition Dp3galloylGa had a positive effect on the value of h. Based on those conclusions we can see increasing TA, Dp3′galloylGa and Qu3acetylGa, the value of a* and C* increased, but L* and b* decreased that means the flower colors change to be red and blue and much vivid. The compounds Chal2′Gal only exist in yellow water lily cultivars. And in the equation of b* the same result is gained: the higher contents of Chal2′Gal, the deeper of the yellow flower color. From the equations, we could see that increasing TA and the content of Qu3acetylGa the flower color would be more vivid.

Comparison of anthocyanin components among cultivars

The main anthocyanins in the petals of tropic water lily were delphinidin glycosides, followed by cyanidin glycosides. Dp3′Ga was presented in all cultivars with blue colors, the highest amounts was detected in cultivar ‘Tai Guo Wang’ which the flower demonstrated blue purple (similar to Fig. 1E). Dp3Ga was only detected in ‘Huang Guan Zi', ‘34’ and ‘Fo Shou Lian’ which ranked the first three cultivars with the highest delphinidin derivates (Table S4, marked by boldface). It can be concluded that at 3-position of the B ring galactose was preferred to be linked, however, in tree peony [10], [11], Lycoris longituba [35] and lotus [36], glucose was abundant at the same position. It would be possibly unique for water lily plants, since flavonoids were thought to be evolutionarily adaptive for plants, the enzyme function for glycosylation of flavonoids might be also evolved in water lily. Within the blue group, Cy3Ga was the only one detected in the petal of ‘Huang Guan Zi’ with the highest TA value. The type of anthocyanins within flowers of ‘18’ (Fig. 1B), ‘27’ (Fig. 1C) and ‘Tai Guo Wang’ (Fig. 1I), the colors of which presented thin-blue, dark-blue and blue purple respectively, maybe due to the increased content of Dp3′Ga or TA value. It could be deduced that Dp3′Ga contributes most of the blue color formation in tropic water lily.

In contrast, in the amaranth group (represented by Fig. 1G), Dp3Ga was the only detected delphinidin derivatives with the highest amount. Compared with blue group, cultivars which were detected Dp3Ga presented amaranth, or detected Dp3′Ga for blue colors. In red group, the derivatives of Dp and Cy were more complicated, Dp3RhGa, Cy3Ga5Rh were also detected. ‘Albert Greenberg’ (Fig. 1D) and ‘Roxburgh’ (Fig. 1H) were illustrated with different colors, which can be deduced by the type of Dp and Cy derivatives and the amount of Cy derivative, the former contained Dp3RhGa, Dp3Ga, the latter with Dp3Ga, Dp3Rh, Dp3′Ga and Cy3Ga, although the content of Dp derivatives was close, the latter had the maximal amount of Cy derivatives.

To the best of our knowledge, besides glycosylation and hydroxylation, acylation also happened to tropic water lily. From Table S4 we can see 2″-galloyl-6″-acetyl presented in most cultivars of tropic water lily, but the highest amount existed in red group. It was obvious to know that 6″-acetyl and 2″-galloyl-6″-oxalyl were only detected in amaranth and red group respectively, but the contents were not high. 2″-galloyl was detected in amaranth, red group and some cultivars of blue group. No anthocyanins were detected within white and yellow group. These two groups might be useful breeding materials when crossing with the other groups to produce novel colors of cultivars.

Putative flavonoid biosynthesis pathway of tropical water lily

Flavonoid especially anthocyanin biosynthesis pathway is one of the most extensively studied pathway of plant secondary products [37]. With the detected component of flavonoids in tropical water lily, we deduced the putative flavonoid biosynthesis pathway relevant to flower color (Fig. 5). It rooted from coumaroyl-CoA and malonyl-CoA, with the enzymes of CHS, CHI and F3H, finally, synthesized dihydrokaempferol. Then it was divided into five sub-pathway for synthesis of anthocyanins and flavonols. With the function of F3′H, F3′5′H, DFR and ANS or FLS, anthocyanidins (cyanidin and delpinidin) and aglycone of flavonols were obtained, which finished the first important modification of hydroxylation of flavonoids. The obtained secondary metabolites were glycosylated by glycosyltransferase at different position in tropical water lily. Flavonoids including anthocyanins exist in glycosylated form in vivo, although most anthocyanins are glycosylated at 3-O-position and often at 5-O-position, the former is a perquisite for further modifications including second glycosylation, acylation and methylation [37]. In tropical water lily, glycosylation at 3-O-position was most often, and the galactose was preferred to be added to the anthocyanidins. The selection of different substrates or different position for glycosylation in different plants may shed light on the related function of glycosyltransferase during the evolution of plants or the enzymes themselves. It is still need to be further studied for the function of this kind of enzymes. Modification of flavonoids by hydroxylation, glycosylation, methylation and acylation played an important role for flower color formation. In tropical water lily, the glycosylated flavonoids were further modified by acyltransferase. It is presumed that acylation with aromatic organic acid to contribute for stabilization of flavonoids due to intra- and/or inter-molecular stacking as co-pigmentation. Most acylated flavonoids in the petals of tropical water lily may intensify blue color as a bathochromic effect. The exact flavonoid biosynthesis pathway relevant to flower color of tropical water lily was still need to be confirmed by more molecular biology or biochemical evidences.

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Figure 5. Putative flavonoid biosynthesis pathway related to flower color of tropical water lily.

The red arrows indicate the biosynthesis pathway of anthocyanins, the dotted arrows indicate uncertain reaction. Cy: cyanidin; Dp: delphinidin; Qu: quercetin; Km: kaempferol; My: myricetin; Is: isorhamnetin; Ga: glactoside; G: glucoside; Rh: rhamnoside; CHS: chalcone synthase; CHI : chalcone isomerase, F3H: flavonoid 3-hydroxylase; F3′H: flavonoid 3′-hydroxylase; F3′5′H: flavonoid 3′,5′-hydroxylase; DFR: dihydroflavonol reductase; FLS: flavonol synthase; ANS: anthocyanidin synthase; GT: flavonoid glycosyltransferase; AT: acyltransferase; MT: methyltransferase; CoA: acetyl coenzyme A.

 

https://doi.org/10.1371/journal.pone.0034335.g005

Discussion

As a perennial plant, tropical water lily is characteristic of some unique biological features such as the special flower colors like blue, violet and bluish purple and some cultivars blooming at night. In general, tropical water lily spreads all over the world and its flower colors are diverse. In this study, using an HPLC-DAD/MS analytical method to characterize flavonoids of water lily petals within 50 min, finally 34 flavonoids have been identified at 525 nm and 350 nm, respectively, in which three anthocyanins, twelve flavonols and one chalcone were discovered for the first time in petals of water lily.

Compared the graphs of HPLC with the results of MLR analysis, we could see that Dp3′galloyl-acetylGa (a7) and Dp3galloyl-acetylGa (a9) were the main anthocyanins in those 35 cultivars, but they did not take part in the MLR analysis and in reverse Dp3′galloylGa (a2) and Dp3galloylGa (a3) replaced them. Meanwhile compared with a2 compound, a7 owned an acetyl group at 6″ position, so did a9. However, there were no similar regularities in flavonols, so the results of MLR analysis were synthetic with anthocyanins, flavonols and chalcones. A correlation analysis indicated that the L* value increased with increasing proportions of Km7galloylGaRh (f10) and Qu3Rh (f11) and the reduction of the contents of a2. At the same time we could see that increasing the contents of TA, Qu3acetylGa (f23) and a2, the value of a* and C* increased while L* and b* decreased, which means that the flower colors changed to be red, blue and much vivid. The compound Chal2′Ga (f13) only existed in yellow water lily cultivars, and in the equation of b*, the same result was gained that the higher contents of f13, the deeper of the yellow flower color. So it was the indispensable compound in formation of the yellow flower in water lily.

The relationships between color parameters and flavonoid compositions showed that many kinds of compounds played an important role on color formation. To characterize the mechanism on flower color formation of special blue and bluish violet which hardy water lily lacks was necessary because it could provide powerful evidences for the ornamental breeding of hardy water lily with blue colors and help to classify cultivars of Nymphaea through phytochemical analysis. At the same time, a HPLC fingerprinting database of water lily cultivars could be established with the flavonoids composition data for discriminating cultivars.

Anthocyanins not only contribute to flower colors, but also play a vital role on bioactivity including anti-oxidant, anti-cancer, anti-allergic and anti-ulcer, therefore they were used as diets in cancer therapy and prevention [38]. Among five anthocyanidins, delphinidin and cyanidin inhibited LPS-induced COX-2 expression, but pelargonidin, peonidin and malvidin did not, the structure-activity relationship suggested that the ortho-dihydroxyphenyl structure of anthocyanidins on the B-ring appears to be related to the inhibitory actions, especially delphinidin as the most potent inhibitor [39]. Delphinidin also protects human HaCaT keratinocytes and mouse skin against UVB-mediated oxidative stress and apoptosis [40]. In tropical water lily, cyanidin and delphinidin are the main anthocyanidins, which may be judged for the high bioactivity for developing functional food or medicine materials.

Other flavonoids were also important components for antioxidant activity, among which quercetin and kaempferol have high antioxidant activity; apigenin and chalcononaringenin or its derivates demonstrated high antioxidant ability due to hydroxylation of B-ring at 4-position [11]. In yellow group of tropical water lily, the content of Chal2′Ga accounted for 80% of the total amount of other flavonoids (TF). However, in blue group the amount of qucercetin was about 87.7% of TF, in amaranth group the amount of kaempferol reached 92.8% of TF. These abundant flavonoids will be good resource for bioactivity for future utilization.

Genes for enzymes involved in the synthesis of flavonoid for flower colors were cloned in model plants such as petunia, arabidopsis, rice etc. People tried to change or create novel flower colors through over-expressed or knocked down some genes related to this pathway [41]. The deduced pathway in this study will help to understand its synthesis of flavonoids and to clone the related genes for molecular breeding of tropical water lily with novel flower colors or studies on molecular mechanism for the formation of flower colors.

Materials and Methods

Standards and solvents

Malvidin 3,5-di-O-glucoside chloride (Mv3G5G) was purchased from Extrasynthese (Genay, France). quercetin 3-O-rutinoside (rutin) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile and methanol used for high-performance liquid chromatography-photodiode array detection (HPLC-DAD) and electrospray ionization multistage mass spectrometry (ESI-MSn) analysis were of chromatographic grade and obtained from Alltech Scientific (Beijing, China). Trifluoroacetic acid (TFA; ≥99%) was purchased from Merck (Darmstadt, Germany). Methanol, formic acid and hydrochloric acid of analytical grade were obtained from Beijing Chemical Works (Beijing, China). HPLC-grade water was purchased from a Milli-Q System (Millipore, Billerica, MA, USA).

Plant Material and Petal Color Measurement

Petals of 35 tropical water lily cultivars were all collected in 2010 at Beijing Botanical Garden, Institute of Botany, the Chinese Academy of Sciences (Long. 39°48′N, Lat. 116°28′E, Alt. 76 m), Beijing, China. These cultivars introduced from all around the world have been planted in the same-sized containers (diameter, 40 cm; height, 30 cm) with two cultivars in each small pond (length×width×depth: 140 cm×140 cm×70 cm) in Beijing Botanical Garden for more than 3 years under the same cultivated conditions like fertilization, irrigation, disease and insect prevention and so on. Petals were harvested at the first blooming day.

The fresh petals were compared to Royal Horticultural Society Colour Chart (RHSCC) and the color parameters were measured with a spectrophotometer NF333 (Nippon Denshoku Industries Co. Ltd., Japan). Then petals from 35 cultivars were stored in refrigerator at −40°C for future analysis. In this study, 35 cultivars were classified into four groups roughly in terms of floral colors (Table 2). CIE 1976 L*a*b* (CIELAB), a software package, is used to measure different aspects of a flower color and takes into account all aspects of the color described by L*, a*, and b* parameters [42]. The L* describes the lightness of the color, ranging from black (L* = 0) to white (L* = 100). The a* negative and positive are for green and red, and the b* negative and positive are for blue and yellow, respectively [35]. The color coordinates measured were shown in Fig. 6: the L* values ranged from 44.36 to 99.92, a* values from −13.40 to 62.91 and b* values from −60.86 to 15.24. Two new parameters, Chroma [C* = (a*2+b*2)1/2] and hue angle [h = arctan b*/a* ], were derived from a* and b*. The Chroma parameter describes the saturation of the color. The C* value is higher, the color is more saturated. The hue angle parameter describes the hue of the color. Hue angle values are stepped counterclockwise across a continuously fading hue circle, some special colors' values of which are remarkable: magenta (0°/360°), yellow (90°), bluish-green (180°) and blue (270°) [42], [43].

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Figure 6. Flower color distribution of Tropical water lily cultivars in coordinate systems of bivariate (a* and b*) (A) and trivariate (a*, b*, and L*) (B), respectively.

The flower colors were identified by the RHSCC value.

 

https://doi.org/10.1371/journal.pone.0034335.g006

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Table 2. Petal colors and color parameters of 35 cultivars of water lily.

 

https://doi.org/10.1371/journal.pone.0034335.t002

Preparation of standard solutions

The standards of Mv3G5G and rutin weighted accurately were dissolved in 0.1% (V/V) HCl-methanol and methanol, respectively, and then diluted to a series of concentrations (mg/mL): 0.01, 0.025, 0.05, 0.1, 0.2, 0.4, 0.6 and 0.8.

Extraction and Preparation of the Flavonoids

The extraction method of flavonoids was modified from that of Yang et al. [36]. Approximately 0.6 g of frozen petal was powdered in liquid nitrogen with mortars and pestles and extracted for the first time with 3 mL 70% (V/V) methanol aqueous solution containing 0.1% HCl shaken in a QL-861 vortex (Kylinbell Lab Instruments, Jiangsu, China), sonicated in KQ-500DE ultrasonic cleaner (Ultrasonic instruments, Jiangsu Kunshan, China) at 20°C for 20 min, centrifuged in SIGMA 3K30 (SIGMA centrifugers, Germany) (12000 rpm, 10 min), and the supernatant was collected. Additional 2 mL and 1 mL extraction solution was supplemented to the residue, and repeated aforesaid operation for second and third times. All extract was pooled and filtrated through 0.22 µm reinforced nylon membrane filters (Shanghai ANPEL, Shanghai, China) before the HPLC-DAD and HPLC-MS analyses. Three replicates were performed for each sample.

HPLC-DAD Systems and Conditions

HPLC analysis was performed on a Dionex (Sunnyvale, CA, USA) system including a P680 HPLC pump, an UltiMate 3000 autosampler, a TCC-100 thermostated column compartment and a PDA100 photodiode array detector. The liquid chromatograph was equipped with an ODS-80Ts QA C18 column (250 mm×4.6 mm i.d., Tosoh, Tokyo, Japan), which was protected with a C18 guard cartridge (Shanghai ANPEL Scientific Instrument, Shanghai, China). Eluent A was 10% formic acid aqueous solution; eluent B was 0.1% formic acid in acetonitrile [44]. A gradient elution as follows was used: 8% B at 0 min, 18% B at 15 min, 23% B at 25 min, 40% B at 45 min, 8% B at 50 min. The flow rate was 0.8 mL·min−1 and aliquots of 10 µL of analytes were injected. Column temperature was maintained at 35°C for all analyses. Chromatograms were acquired at 520 and 350 nm for anthocyanins and other flavonoids, respectively, and DAD data were recorded from 200 to 800 nm.

HPLC-MS System and Conditions

HPLC-ESI-MSn analysis for anthocyanins and other flavonoids were carried out in an Agilent-1100 HPLC system equipped with a UV detector and a LC-MSD Trap VL ion-trap mass spectrometer via an ESI source (Agilent Technologies, Palo Alto, CA, USA). The HPLC separation conditions were the same as mentioned above. The MS conditions were as follows: anthocyanins were adopted in positive-ion (PI) mode and other flavonoids were employed in negative-ion (NI) mode. ESI was performed by using the following conditions: capillary voltage, 4.0 kV; a nebulization pressure, 241.3 kPa; and a gas (N2) temperature, 350°C; flow rate, 8.0 L·min−1. Capillary offset and exit voltage were 77.2 V and 127.3 V, respectively for PI, and −77.2 V and −127.3 V separately for NI. MS spectrum was recorded over the range from m/z 100 to 1000.

Statistical Analysis

Correlations between petal color parameters and flavonoid compositions of individual varieties were analyzed by a multiple linear regression (MLR) (SPSS17.0 Inc., CHI, IL, USA) with stepwise method.

Supporting Information

Relationship between the Composition of Flavonoids and Flower Colors Variation in Tropical Water Lily (Nymphaea) Cultivars

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Figure S1.

The graphs of glycosides of flavonoids (350 nm) separated between longer column (250 mm) (a) and shorter column (150 mm) (b).

https://doi.org/10.1371/journal.pone.0034335.s001

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Table S1.

Linearity of response for Mv3G5G and rutin using the optimized method. Calibration fitting: y = kx+m1. 1 In the regression equation y = kx+m, y refers to the peak area, x is concentration of the standard substances (µg/mL), r2 is the correlation coefficient of the equation.

https://doi.org/10.1371/journal.pone.0034335.s002

(DOC)

Table S2.

Intra- and inter-day precision of 31 main flavonoids in the extract of water lily petals by HPLC-DAD.

https://doi.org/10.1371/journal.pone.0034335.s003

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Table S3.

HPLC-DAD and HPLC-ESI-MSn analysis as well as the structure characterization and tentative identification of glycosides of flavonol and chalcone in petals of water lily.

https://doi.org/10.1371/journal.pone.0034335.s004

(DOC)

Table S4.

The mean content (µg/g) of petal anthocyanin in 35 tropic water lily varieties.

https://doi.org/10.1371/journal.pone.0034335.s005

(DOC)

Acknowledgments

We would like to thank Chonghui Li, Hui Du, Jie Wu, Xia Jiang and Shanshan Li for their help in collecting plant materials and amending Fig. 6 and Fig. 5.

Author Contributions

Conceived and designed the experiments: L-SW Q-YS. Performed the experiments: M-LZ Y-JX HL X-CZ L-JW. Analyzed the data: M-LZ X-CZ. Contributed reagents/materials/analysis tools: M-LZ H-JZ HL P-XZ. Wrote the paper: M-LZ QY-S.

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