J Biosci (2021)46:3 Ó Indian Academy of Sciences

DOI: 10.1007/s12038-020-00125-3 (0123456789().,-volV)(0123456789().,-volV)

Metabolite profiling of violet, white and pink flowers revealing flavonoids composition patterns in Rhododendron pulchrum Sweet

1 1 1 1 SHUZHEN WANG ,SHIYING HUANG ,JIE YANG ,ZHILIANG LI , 1 1 2 MINGJU ZHANG ,YUANPING FANG ,QIAOFENG YANG * and 1 WEIBIN JIN * 1Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization, Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains, Hubei Zhongke Research Institute of Industrial Technology, and College of Life Science, Huanggang Normal University, Huanggang 438000, Hubei Province, China 2College of Food and Bioengineering, Henan University of Animal Husbandry and Ecomomy, Zhengzhou 450000, Henan Province, China

*Corresponding authors (Emails, [email protected]; [email protected]) MS received 17 June 2020; accepted 27 November 2020

Flower color is the major characteristics and critical breeding program for most Rhododendron species. However, little is known about their coloration mechanism and color inheritance. In this study, petal pigment constituents of three Rhododendron pulchrum Sweet cultivars with different colors were clarified based on LC- ESI-MS/MS method. Using a broad-targeted metabolomic approach, a total of 149 flavonoids and their glycosylated or methylated derivatives were identified, including 18 (Pg, Cy, Dp, Pn, Pt, and Mv) and 32 flavonols (mainly 3-O-glycosides and 3-O-glycosides). Moreover, anthocyanins were mainly represented by -3-O-glycosides (glucoside, rutinoside, galactoside, and di-glyco- sides). Flavone and C-glycosylated flavone were major second metabolites responsible for the difference among three different R. pulchrum cultivars. The accumulation of total flavonoids displayed a clear phenotypic variation: cultivars ‘zihe’ and ‘fenhe’ were clustered together, while ‘baihe’ was clustered alone in the HCA analysis. The composition and content of anthocyanins were more complex in colored flowers (‘zehe’ and ‘fenhe’) than in white flower (‘baihe’). This study further enhanced our understanding on the flavonoids profile of flower coloration and will provide biochemical basis for further genetic breeding in Rhododendron species.

Keywords. ; flower color; genetic breeding; metabolomics; Rhododendron pulchrum Sweet

1. Introduction flavonoids, carotenoids, and (Liu et al. 2016). , belonging to the class of phenyl- Flower color is one of the most important breeding propanoids, provide a wide range of colors ranging program objectives in ornamental plants (Liu et al. from pale-yellow to blue (Hang et al. 2011). Flavo- 2016). Many factors, such as pigment composition, pH, noids are widely distributed in many flowers, leaves, cell shape, and co-pigmentation, are related to petal seeds and fruits, which also possess healthy benefits, colors (Hang et al. 2011; Sinopoli et al. 2019). Among such as protecting against certain cancers and inflam- these factors, pigment composition is the most impor- matory disease (Sinopoli et al. 2019; Herna´ndez et al. tant, as quality trait of flower color is largely deter- 2009). Depending on their structures, flavonoids could mined by metabolic composition, especially be classified into six types, including chalcones, fla- Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12038-020-00125-3) contains supplementary material, which is available to authorized users. http://www.ias.ac.in/jbiosci 3 Page 2 of 10 Shuzhen Wang et al. vones, flavonols, flavanones, flavan-3-ols, and antho- identified in Rhododendron species (Park et al. 2018; cyanins (Veitch and Grayer 2011; Dong et al. 2014; Du et al. 2018). The red petals contain 2–4 major Tanaka et al. 2008). anthocyanins, as 3-galactoside and cyanidin Anthocyanins biosynthesis is an important part of 3-arabinoside are common to Rhododendron simsii flavonoid branch within the pathway with red flowers (Park et al. 2018). However, pigments (Aderson et al. 2015; Dong et al. 2019; Kim et al. composition in petals of azalea species is limited. 2017; Rothenberg et al. 2019; Wang et al. 2018a). As Metabolomics aims to analyze all metabolites in a anthocyanidin glycosides, anthocyanins play crucial biological sample comprehensively, and shows great roles in protecting plant against stress conditions, oxi- potential in elucidating plant metabolic processes. In dant damage, and infection by pathogens (Rothenberg this study, the pigment compositions of three R. pul- et al. 2019). In particular, six are chrum cultivars with different colors were analyzed common in vascular plants, including using LC-ESI-MS/MS, aiming at clarifying the features (Pg), (Pn), (Pt), cyanidin (Cy), of pigment composition, as well as providing basis for (Mv), and (Dp) (Zhang et al. genetic improvement of flower color in R. pulchrum 2014). Moreover, anthocyanins derivatives Dp, Pt, and and other Rhododendron species. Mv are source of violet and dark colors, whereas the derivatives Cy and Pg are main pigments in bright-red- color fruits (Cho et al. 2016). In particular, Mv 3-O- 2. Materials and methods arabinoside-5-O-glucosed and DP 3-O-arabinoside-5- O-glucosed play important roles in violet coloration 2.1 Plant materials (Park et al. 2018). Up to now, more than 635 antho- cyanins and 23 anthocyanidins have been well identi- R. pulchrum cultivars ‘fenhe’, ‘baihe’, and ‘zihe’ with fied in fruits, vegetables, and flowers (Castan˜eda et al. pink, white, and violet flowers were sampled from 2009). Huanggang Normal University Botanical Garden The genus Rhododendron, including approximately (30°1601200N, 114°3101200E, 200–248 m) (figure 1). 1000 species and most unique to China, was introduced Colors of R. pulchrum petals were evaluated according into Europe at the end of the 18th century (Popescu and to the Royal Horticultural Society Colour Kopp 2013; Wang et al. 2017). A great many of Chart (RHSCC) under natural light. For each specific Rhododendron species, such as R. simsii, R. molle, and color, petals were harvested randomly from six indi- R. fortunei, have valuable horticultural and medicinal viduals and pooled for each biological replicate. All values, in addition to the beautiful vegetative forms and samples were harvested on the same day, immediately bright-colored flowers (Wang et al. 2017). As most placed in liquid nitrogen and stored at -70°C until valuable and largest genera of woody plants, Rhodo- vacuum freeze-drying. dendron was first used as ornamental plants in Eng- land, and then developed in Belgium and Holland. In particular, various flower colors were observed in 2.2 Chemicals Rhododendron, such as red, red-purple, purple-violet, purple, purplish pink, white, yellow, and pink, which All the chemicals were of analytical reagent grade. show high aesthetic and economic values (Hang et al. HPLCt-grade methanol, acetonitrile, and acetic acid 2011). In addition, blotches, stripes, and even white were purchased from Merck Company (Germany). The margin were observed in petals of some Rhododendron water was doubly purified with a MilliQ ULTRA water species (Du et al. 2018; Mizuta et al. 2009). purification system (Millipore, Bedford, MA). Anthocyanin and flavonols, such as Cy, Dp, Mv and Authentic standards were brought from ANPEL their glycoside derivatives, are also major pigments in (Shanghai, China) and Sigma-Aldrich (USA). Stan- the genus Rhododendron, which have been isolated dards stock solutions were prepared in methanol or using paper chromatography, thin-layer chromatogra- methanol-dimethyl sulfoxide (DMSO, 50:50, v/v), and phy (TLC), and high-performance liquid chromatog- stored at -20°C in darkness. The internal standard was raphy (HPLC) (Sinopoli et al. 2019;Duet al. 2018). combined standard solutions of flavonoids, which were Moreover, their structures were elucidated by 1H-NMR prepared just before use by mixing individual stock spectroscopic (Sinopoli et al. 2019). Quercetin, myr- solutions and diluted with 70% aqueous methanol. icetin, mearnsetin, kaempferol, isorhamnetin, dios- Quality control samples (QC) were prepared from a metin and their glycoside derivatives all have been combined sample by mixing all sample extracts, then Metabolite profiling of Rhododendron pulchrum Sweet Page 3 of 10 3

Figure 1. R. pulchrum flower tissues sampled for metabolomics study of flavonoids: (A) cultivar ‘zihe’, violet flower; (B) cultivar ‘fenhe’, pink flower; (C) cultivar ‘baihe’, white flower. were divided into three equal parts (mix 1, mix 2, and 0 min, 5:95 V/V at 10.0 min, 5:95 V/V at 11.0 min, mix 3). During the experiments, QC were injected 95:5 V/V at 11.1 min, and 95:5 V/V at 15.0 min). The every five samples to assess the repeatability. effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (Q TRAP)-MS. The ESI source operation parameters were as follows: ion 2.3 Sample preparation, extraction, and LC-MS source, turbo spray; source temperature, 550°C; ion analysis spray voltage (IS) 5500 V; ion source gas I (GSI), 55 psi; gas II (GSII), 60 psi; curtain gas (CUR), 25.0 psi; The freeze-dried petals were ground using a mixer mill and the collision gas (CAD) was high. Instrument (MM 400, Retsch) with a zirconia beads for 1.5 min at tuning and mass calibration were performed with 10 30 Hz. Then, a 100 mg mass of powder was weighted and 100 lmol/L polypropylene glycol solutions in and extracted overnight at 4°C with 1.0 ml of 70% QQQ and LIT modes, respectively. The QQQ scans aqueous methanol. Following centrifugation at were acquired as MRM experiments with the collision 10,000g for 10 min, the extracts were filtered (SCAA- gas (nitrogen) being set to 5 psi. The DP and CE for 104, 0.22 lm pore size; ANPEL, Shanghai, China, individual MRM transitions were performed with fur- http://www.anpel.com.cn/), and then were analyzed ther DP and CE optimization. A specific set of MRM using an HPLC-ESI-QTRAP-MS/MS system (HPLC, transitions was monitored for each period according to Shim-pack UFLC SHIMADZU CBM30A system, the metabolites that were eluted within this period. www.shimadzu.com.cn/; MS, Applied Biosystems 4500 Q TRAP, www.appliedbiosystems.com.cn/) (Chen et al. 2013). The data recorded was controlled by 2.4 Identification of metabolites and statistical MassHunter Qualitative Analysis software (Agilent analysis Technologies, Barcelona, Spain). Quantification of metabolites was performed in the multiple reaction Qualitative analysis of metabolite was based on the monitoring (MRM) model. Data acquisition, peak MVDB V3.0 Database of Wuhan Metware Biotech- integration and calculations were performed with nology Co., Ltd. (Wuhan, China). Compounds pos- Analyst 1.6.2 software (AB Sciex). LIT and triple sessing standards was identified through comparison of quadrupole (QQQ) scans were acquired on a triple the accurate m/z values, retention time (RT), and quadrupole-linear ion trap mass spectrometer (Q fragmentation patterns with those standards. For TRAP) using an API 4500 Q TRAP LC/MS/MS Sys- metabolites without standards, fragmentation patterns tem, which was equipped with an ESI Turbo Ion-Spray similar to the identified metabolites were used to query interface operated in a positive ion mode. the MS2 spectral data taken from the literature or to Chromatographic analysis was performed using a search MassBank database. The log2 transformed Waters ACQUITY UPLC HSS T3 C18 (1.8 lm, metabolite data were subjected to Principal Component 2.1 mm 9 100 mm) with a column temperature of Analysis (PCA). The ‘heatmap.2’ function in the 40°C. A 5 lL volume of extract was injected, and flow ‘gplot’ R-package (http://cran.r-project.org/web/ rate was set as 0.35 mL/min. The solvent system packages/gplots/index.html) was utilized in Hierarchi- consisted of solvent A (water with 0.04% acetic acid) cal Clustering Analysis (HCA). Metabolite data was and solvent B (acetonitrile with 0.04% acetic acid), as firstly log2 transformed to improve the normality, fol- well as a gradient program (solvent A/B: 100:0 V/V at lowed by a min-max normalization for HCA. The VIP 3 Page 4 of 10 Shuzhen Wang et al. Table 1. Anthocyanins identified in the pestals of R. pulchrum cultivars

Ion Retention Molecular Ionization KEGG Compound mode Q1 (Da) Q3 (Da) time (min) Weight (Da) model ID Peonidin O-hexoside Positive 4.63E?02 3.01E?02 3.00 4.63E?02 Protonated - O-hexoside Positive 4.77E?02 3.16E?02 3.22 4.77E?02 Protonated - Cyanidin 3-O-glucoside Positive 4.49E?02 2.87E?02 2.45 4.49E?02 Protonated C08604 (Kuromanin) Delphinidin O-malonyl- Positive 6.37E?02 3.04E?02 3.16 6.37E?02 Protonated - malonylhexoside Peonidin Positive 3.01E?02 2.74E?02 3.86 3.01E?02 Protonated C08726 Cyanidin O-syringic acid Negative 4.65E?02 2.85E?02 2.41 4.66E?02 [M-H]- - Peonidin O-malonylhexoside Negative 5.47E?02 5.03E?02 2.87 5.48E?02 [M-H]- - Delphinidin Positive 3.03E?02 1.49E?02 2.90 3.03E?02 Protonated C05908 Malvidin 3-O-galactoside Positive 4.93E?02 3.32E?02 2.81 4.93E?02 Protonated - Malvidin 3-O-glucoside () Positive 4.93E?02 3.32E?02 2.86 4.93E?02 Protonated C12140 Delphinidin 3-O-glucoside Positive 4.65E?02 3.03E?02 2.30 4.65E?02 Protonated C12138 (Mirtillin) Cyanidin 3-O-rutinoside Positive 5.95E?02 2.88E?02 2.60 5.95E?02 Protonated C08620 (Keracyanin) Cyanidin 3,5-O-diglucoside Positive 6.11E?02 2.88E?02 2.26 6.11E?02 Protonated C08639 (Cyanin) Positive 5.95E?02 2.72E?02 2.45 5.95E?02 Protonated C08725 Delphinidin 3-O-rutinoside Positive 6.11E?02 3.03E?02 2.26 6.11E?02 Protonated C16315 () Petunidin 3-O-glucoside Positive 4.79E?02 3.17E?02 2.42 4.79E?02 Protonated C12139 Pelargonidin 3-O-beta-D- Positive 4.33E?02 2.71E?02 2.58 4.33E?02 Protonated - glucoside( chloride) Cyanidin Positive 2.87E?02 2.32E?02 3.45 2.87E?02 Protonated C05905

values (Variable Importance for the Projection [1) and isoflavone, 18 anthocyanins, 3 procyanidins, and 1 FC scores (fold-change C2orB0.5) were used to flavonolignan (Supplementary Table 1). In addition, identify differential accumulation of metabolites flavone 5/7-O-glycosides and C-glycosylated flavone between cultivars. One-way ANOVA (pB0.05) was were also the major flavonoid constituents in R. pul- also performed. Furthermore, PCA and partial least chrum. Six major anthocyanidins, including Pg, Cy, squares-discriminate analysis (PLS-DA) were per- Dp, Pn, Pt, and Mv, were all detected. Moreover, formed with SIMCA-P 14.0 software package. anthocyanins were mainly represented by anthocyani- din-3-O-glycosides (glucoside, rutinoside, galactoside, and di-glycosides). Apart from the widespread form of 3. Results anthocyanidin-O-glycosides, some anthocyanidins were even acylated by malonyl (table 1). Furthermore, 3.1 Pigment identification in R. pulchrum petals kaempferol 3-O-glycosides and quercetin 3-O-glyco- sides were the main types of flavonols accumulated in The RHSCC numbers of the petals of three R. pul- R. pulchrum petals. chrum cultivars (‘zihe’, ‘fenhe’, and ‘baihe’) were classified in violet, pink, and white group, respectively. Chromatographic analysis showed that second 3.2 Cluster analysis of R. pulchrum cultivars metabolites flavonoids were major pigments in R. based on flavonoids profiles pulchrum flower, especially flavonols and antho- cyanins. Totally, 149 known flavonoids and their gly- According to the metabolic profile, cultivars ‘zihe’ and cosylated or methylated derivatives were obtained, ‘fenhe’ were clustered together, while ‘baihe’ was including 38 flavone, 32 flavonol, 27 flavone C-gly- clustered alone (figure 2A). Accumulation of flavo- cosides, 9 derivatives, 14 flavanone, 7 noids displayed a clear phenotypic variation in Metabolite profiling of Rhododendron pulchrum Sweet Page 5 of 10 3

Figure 2. Heatmap visualization (A) and Principal component analysis (B) of detected metabolites in three cultivars ‘zihe’, ‘fenhe’, and ‘baihe’. cultivars with different colored flowers. The signal separated cultivars ‘fenhe’ and ‘baihe’ from ‘zihe’ intensity produced from MS by all metabolites was successfully. In addition, the second component (PC 2, relative to its abundance. Based on the species-specific 31%) separated cultivars ‘fenhe’ and ‘zihe’ from cul- accumulation patterns, flavonoid could be clearly tivar ‘baihe’. Moreover, metabolite composition of grouped into two main clusters with six sub-clusters ‘zihe’ and ‘fenhe’ were more similar when compared (figure 2A). In cluster I, the main flavonoids was with ‘baihe’. The closer relationship between cultivars anthocyanins, flavone, and flavone C-glycosides, ‘zihe’ and ‘fenhe’ might be caused by smaller meta- including Dp 3-O-rutinoside, Mv 3-O-galactoside, bolic variations. chrysoeriol O-glycosides, C-glycosylated-hesperetin and O-glycosides, which had higher levels in cultivar ‘zihe’ than ‘fenhe’ and ‘baihe’. In cluster II, the 3.3 Differences of pigment accumulation patterns dominant flavonoids were anthocyanidin, flavonol, in different cultivars catechin and its diversity, flavone and flavone C-gly- cosides, such as cyanidin, peonidin delphinidin, To identify metabolites responsible for the observed kaempferol 3-O-rhamnoside, quercetin 40-O-glucoside, color differences, pairwise comparisons and PLS-DA and luteoin C-glycosides. The differential accumulation were carried out. Obvious clustering in each group and of flavonoid between cultivars ‘fenhe’ and ‘baihe’ significant differences of flavonoids between cultivars further divided cluster II into three subclusters (II-a, II- ‘zihe’ and ‘baihe’, as well as between cultivars ‘fenhe’ b, II-c). In cluster II-c, flavonoids (7 catechin and its and ‘baihe’ were obtained. Furthermore, the differences derivatives, 18 flavonols) displayed highest levels in R. between ‘zihe’ and ‘fenhe’ was relatively smaller. pulchrum cultivar ‘baihe’. Goodness of fit (R2X[1] = 0.925, R2X[2] = 0.0409, Multivariates statistics were used to explore the dif- and R2Y = 1) and predictability (Q2 = 1) of the pattern ferential accumulation of metabolites among different analysis were observed, suggesting that the model was cultivars (figure 2B). Based on metabolite profile, the clearly classified and predictable (supplementary Pearson correlation coefficient among different samples figure 1). were calculated, and results showed that the biological According to the FC scores and VIP values set replicates in each sample had high correlation. PCA during data analysis, a great number of differential could separate these R. pulchrum cultivars based on metabolites were obtained based on differential metabolite composition, inferring that color variation metabolite screening (figure 3; supplementary might be strongly influenced by metabolite profiles. In tables 2–4). Compared between metabolic profiling of related to PCA results, the first component (PC1, 40%) cultivars ‘zihe’ and ‘fenhe’, 39 differential metabolites 3 Page 6 of 10 Shuzhen Wang et al.

Figure 3. Numbers of different flavonoids in R. pulchrum cultivars with different flower color through pairwise comparisons (A), and volcano plot of differences between ‘zihe’ and ‘fenhe’ (B), ‘fenhe’ and ‘baihe’ (C), ‘zihe’ and ‘baihe’ (D). The red dot represents up-regulated metabolite (with a fold change C 2, VIP C 1); the green dot represents down- regulated metabolite (with a fold change B 0.5, VIP C 1). Note: DCMs were abbreviation for differential metabolites. were obtained: 25 were up-regulated, 14 were down- Levels of anthocyanins (Delphinidin 3-O-rutinoside), regulated in cultivar ‘zihe’ (figure 3A and B). More- flavone (chrysoeriol O-glycoside and Tricin O-gly- over, 37 (30 down-regulated and 7 up-regulated) and coside) and C-glycosylated chrysoeriol, were higher in 35 (27 down-regulated and 8 up-regulated) differential cultivar ‘zihe’ than cultivar ‘fenhe’. Meanwhile, metabolites were found in cultivar ‘baihe’ when com- amount of flavonol was higher in cultivar ‘fenhe’ than pared with cultivars ‘zihe’ and ‘fenhe’, respectively cultivar ‘zihe’. In particular, the content was higher in (figure 3C and D). purple flower than that in pink flower, including del- Compared with cultivar ‘baihe’, most anthocyanins phinidin 3-O-glucoside (Dp 3-O-Glu), malvidin 3-O- and flavonoids are up-regulated in both cultivars ‘zihe’ galactoside (Mv 3-O-Gal), malvidin 3-O-glucoside and ‘fenhe’, mainly as cyanidin 3-O-glucoside (Cy (Mv 3-O-Glu), rosinidin O-hexoside (Ros O-Glu), 5-O- 3-O-Glu), cyanidin O-syringic acid (Cy O-syr), del- hexosyl-O-hexoside (Tri 5-O-Hex-O-Hex), tricin 7-O- phinidin 3-O-glucoside (Dp 3-O-Glu), malvidin 3-O- hexosyl-O-hexoside (Tri 7-O-Hex-O-Hex), and hes- galactoside (Mv 3-O-Gal), malvidin 3-O-glucoside peretin C-hexoside O-hexoside (Hes C-Hex-O-Hex) (Mv 3-O-Glu), rosinidin O-hexoside (Ros O-Glu), tri- (figure 4A and B). However, ‘fenhe’ accumulated cin 5-O-hexosyl-O-hexoside (Tri 5-O-Hex-O-Hex), higher levels of cyanidin 3-O-glucoside (Cy 3-O-Glu), tricin 7-O-hexosyl-O-hexoside (Tri 7-O-Hex-O-Hex), di-O-methylquercetin (Di-O-mel-Que), quercetin 5-O- quercetin 5-O-malonylhexosyl-hexoside (Que 5-O- malonylhexosyl-hexoside (Que 5-O-mal-Hex-Hex), mal-Hex-Hex), hexosyl-isorhamnetin O-hexoside (C- and quercetin O-acetylhexoside (Que O-Ace-Hex) Hex-Iso O-Hex), chrysoeriol 6-C-pentosyl-O-ruti- (figure 4A and B). noside (Chr 6-C-Penl-O-Rut), hesperetin C-hexoside In particular, the composition and content of antho- O-hexoside (Hes C-Hex-O-Hex) (figure 4A and B). cyanins were less complex in cultivar ‘baihe’ with However, the amount of Di-O-mel-Que was higher in white flowers than in other colored flowers. In cultivar cultiver ‘fenhe’ than the other two cultivars. Mean- ‘baihe’, 6-C-hexosyl-8-C-hexosyl-O-hexoside while, difference in amount of myricetin was the least showed the highest expression than that in the other in three cultivars with different colored flowers (fig- two cultivars. Furthermore, the amount of C-glycosy- ure 4A and B). lated luteolin (6-C-hexosyl-luteolin O-hexoside, C- Metabolite profiling of Rhododendron pulchrum Sweet Page 7 of 10 3

Figure 4. The different accumulation of metabolites among cultivars ‘zihe’, ‘fenhe’, and ‘baihe’. (A) Change rate of 6 anthocyanins and 2 Trictin-O-glycoside in‘zihe’, ‘fenhe’, and ‘baihe’ cultivars. (B) Rate of change of 3 flavonols and 4 C- glycosided flavone three cultivars. (C) The Venn diagram of metabolic profile between three R. pulchrum cultivars. The numbers show the overlapping and color-specific differential metabolites. (D) Heatmap visualization of the differential metabolites in R. pulchrum flower. hexosyl-luteolin O-hexosyl-O-pentoside, and C-hexo- ‘fenhe’, ‘zihe’ vs ‘baihe’, and ‘fenhe’ vs ‘baihe’, syl-luteolin O-hexoside) was higher than that in culti- respectively (figure 4C). HCA results showed that var ‘zihe’. In particular, 11 anthocyanins, including expression levels of most differential metabolites were delphinidin 3-O-glucoside, delphinidin 3-O- rutinoside, higher in violet flower (cultivar ‘zihe’) compared with malvidin 3-O-glucoside, malvidin 3-O-galactoside, the other two samples (cultivars ‘fenhe’ and ‘baihe’). cyanidin O-syringic acid, cyanidin 3-O-glucoside, Cyanidin 3,5- O-diglucoside, pelargonidin 3-O-beta-D- glucoside, pelargonin, peonidin O-hexoside, and 4. Discussion rosinidin O-hexoside, elevated more than 8-fold in cultivar ‘zihe’ than cultivar ‘baihe’. In related to fla- Flavonoids, one group of the most widespread plant vone, cultivar ‘baihe’ accumulated higher levels of C- second metabolites, are involved in many physiological glycosylated Luteolin, while cultivar ‘fenhe’ accumu- processes, such as pigmentation for fruits and flowers, lated significant higher levels of Chrysoeriol O-gly- protecting plant from ultraviolet-B damage, and phar- coside, Tricin 5-O-hexoside, and Tricin O-glycoside. maceuticals activities (Monti et al. 2016). Rhododen- Among the differential metabolites, 11 metabolites dron species are widely cultivated around the world had been obtained from independent pairwise com- due to their flowers and high medicinal values. It is parisons (‘zihe’ vs ‘fenhe’, ‘zihe’ vs ‘baihe’, and important to clarify pigment composition of the widely ‘fenhe’ vs ‘baihe’), including 4 anthocyanins, 1 flavo- cultivated R. pulchrum, as well as genetic improvement nol, and 6 glycosylated-flavone. Specifically, 13, 7 and of flower color in R. pulchrum, though little is known. 6 differential metabolites existed solely in ‘zihe’ vs In the PCA plot, QC samples were grouped together, as 3 Page 8 of 10 Shuzhen Wang et al. these QC samples had the same metabolic profiles. nyingchiense, and R. oreotrephes (Liu et al. 2016; Moreover, the experimental methods and analysis were Stracke et al. 2007). However, cyanidin 3-O-arabi- quite stable and repeatable. noside existed in R. faucium, R. hirtipes, R. vellereum, In this study, 149 flavonoids with standard annota- R. aganniphum var. Schizopeplum, R. nyingchiense, tion, including 18 anthocyanins and 32 flavonols, have and R. triflorums, but was absent in R. pulchrum (Liu been obtained from R. pulchrum. In 30 Rhododendron et al. 2016). In particular, malvidin 3-O-galactoside species, only 7 anthocyanins and 23 flavonol com- and malvidin 3-O-glucoside were unique to R. pul- pounds have been identified from 30 Rhododendron chrum compared with other Rhododendron species. species investigated by Du et al.(2018). Only 5 Malvidin 3-O-rhamnoside-5-O-glucoside, found in R. anthocyanins and 23 flavonols have been found from nivale, was also absent in R. pulchrum. Similarly, 10 Rhododendron species, including R. virgatum, R. malvidin 3-O-arabinoside-5-O-glucoside was not faucium, R. hirtipes, R. vellereum, R. aganniphum var. detected in R. pulchrum, but was important in R. vir- Schizopeplum, R. nyingchiense, R. nivale, R. oreo- gatum, R. nivale and R. oreotrephes (Liu et al. 2016). trephes, R. wardii, and R. triflorum (Liu et al. 2016). Anthocyanin displayed higher expression levels in Anthocyanins and flavonols were the major pigments violet R. pulchrum, while anthocyanidin were higher in in R. pulchrum petals, in accordance with other pink and white flowers. Purple-violet flowers and Rhododendron species (Mizuta et al. 2009). In addi- purple flowers contained mainly di-O-glycosides of tion, 38 flavones and 27 flavone-glycosides (tricin, malvidin, and less or none cyanidin 3-O-glucoside-5- luteolin, chrysoeriol, and apigenin) have been found in O-arabinoside, while red flowers were mainly formed dicotyledon plant R. pulchrum, but were absent in by cyanidin and peonidin (Liu et al. 2016). Antho- Arabidopsis, as monocotyledon plants lack or had low cyanin constitution of violet flowersvaried larger than activity of flavone synthase (FNS) (Stracke et al. that of red flowers (Zhang et al. 2014). Here, malvidin 2007). In particular, unigene homologous to FNS has 3-O-galactoside and malvidin 3-O-glucoside in violet been found from R. pulchrum in our transcriptome flower showed more than 100-fold higher than that in analysis (Wang et al. 2018b). white flower (cultivar ‘baihe’). However, di-O-glyco- Anthocyanins, containing an anthocyandins back- sides of malvidin were primary in R. nivale (purple- bone (aglycones groups) and glycosyl groups, are the violet flower) and R. oreotrephes (purple flower) (Liu most important pigments of the vascular plans, and et al. 2016). Cyanidin 3-O-rutinoside showed 6-fold were fundamentally important in determining flower higher in pink flower (cultivar ‘fenhe’) than that in color in Rhododendron species (Mizuta et al. 2009). violet flower (cultivar ‘zihe’). Similarly, cyanidin Different flower colors were due to the differences in derivatives were the major anthocyanin in R. triflorum the compositions of aglycones and glycosyl groups and other red-purple flowers (Liu et al. 2016). Antho- (Liu et al. 2016). In particular, most anthocyanins were cyanins could not be detected in white group petals, detected under positive mode, while cyanidin O-sy- although anthocyanins peaks were detected (Liu et al. ringic acid and peonidin O-malonylhexoside were 2016). However, anthocyanins were also detected in detected under negative mode. Specifically, Cy 3-O- white flower, such as delphinidin and cyanidin 3-O- rutinoside, Cy O-syringic acid, Dp 3-O-rutinoside, Mv rutinoside. 3-O-glucoside, Pt 3-O-glucoside, and Pn O-hexoside Flavone and C-glycosylated flavone were major were identified for the first time in Rhododendron second metabolites responsible for the difference species. In particular, except for cyanidin, malvidin, among three different R. pulchrum cultivars. Flavone and delphinidin, other three major anthocyanidins and derivatives might possess protective effect against (pelargonidin, peonidin, and petunidin) and glycosides UV-B-mediated oxidative damage in R. pulchrum,as compounds have also been identified. R. pulchrum also flavones were involved in protecting the plant from accumulates kaempferol 3-O-glycosides and quercetin UV-B damage (Hang et al. 2011; Herna´ndez et al. 3-O-glycosides, which is similar to Arabidopsis 2009). Most flavone exhibited higher in colored flower, (Stracke et al. 2007). Furthermore, Cy-3-O-glucoside including tricin 7-O-hexosyl-O-hexoside, chrysoeriol was found in R. pulchrum, which was also found in R. 6-C-pentosyl-O-rutinoside, hesperetin C-hexoside O- faucium, R. hirtipes, R. vellereum, R. aganniphum var. hexoside, C-hexosyl- isorhamnetin O-hexoside, which Schizopeplum, R. nyingchiense, and R. triflorums (Liu had higher content in violet cultivar than in pink cul- et al. 2016). Moreover, Cy 3,5-O-diglucoside was tivar. Meanwhile, tricin O-glycosylated, and C-glyco- shared by R. pulchrum, R. virgatum, R. faucium, R. sylated-chrysoeriol displayed higher accumulation in hirtipes, R. aganniphum var. Schizopeplum, R. violet flower, while C-glycosylated-luteolin and C- Metabolite profiling of Rhododendron pulchrum Sweet Page 9 of 10 3 glycosylated-apigenin showed higher content in white Boulton R 2001 The of anthocyanins and its flower. role in the color ofred wine: a critical review. Am. J. Enol. 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Corresponding editor: MANCHIKATLA VENKAT RAJAM