Comparative Analysis of Flavonoids and Polar Metabolite Profiling of

Mar 3, 2014 - FeCHI and FeFLS2 genes also showed higher expression levels in seeds of the high-rutin cultivar. In contrast, FePAL, FeC4H, Fe4CL1, FeCH...
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Comparative Analysis of Flavonoids and Polar Metabolite Profiling of Tanno-Original and Tanno-High Rutin Buckwheat Xiaohua Li,†,§ Jae Kwang Kim,‡,§ Soo-Yun Park,∥ Shicheng Zhao,† Yeon Bok Kim,† Sanghyun Lee,⊥ and Sang Un Park*,† †

Department of Crop Science, Chungnam National University, Daejeon 305-764, Korea Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 406-772, Korea ∥ National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Korea ⊥ Department of Integrative Plant Science, Chung-Ang University, Anseong 456-756, Korea ‡

S Supporting Information *

ABSTRACT: Rutin is an important indicator for evaluating the quality of buckwheat. In this study, flavonoid biosynthesis was compared between two common cultivars (an original and a high-rutin line) of buckwheat, Fagopyrum esculentum Moench. Transcriptional levels of the main flavonoid biosynthetic genes were analyzed by real-time PCR, and main flavonoid metabolites were detected by high-performance liquid chromatography (HPLC); levels of gene expression varied among organs of the two cultivars. Significantly higher transcription levels of most flavonoid biosynthetic genes, except FeFLS1, were detected in stems of the high-rutin line than in stems of the original line. FeCHI and FeFLS2 genes also showed higher expression levels in seeds of the high-rutin cultivar. In contrast, FePAL, FeC4H, Fe4CL1, FeCHS, FeF3H, FeF3′H, FeFLS2, and FeDFR were highly detected in the roots of the original line. The HPLC results indicated 1.73-, 1.62-, and 1.77-fold higher accumulation of rutin (the primary flavonoid compound) in leaves, stems, and mature seeds of the high-rutin cultivar (24.86, 1.46, and 1.36 μg/mg, respectively) compared with the original cultivar (14.40, 0.90, and 0.77 μg/mg, respectively). A total of 46 metabolites were identified from seeds by gas chromatography−time-of-flight mass spectrometry. The metabolite profiles were subjected to principal component analysis (PCA). PCA could clearly differentiate the original and high-rutin cultivars. Our results indicate that the high-rutin cultivar could be an excellent alternative for buckwheat culture, and we provide useful information for obtaining this cultivar. KEYWORDS: Fagopyrum esculentum, flavonoid biosynthetic pathway, gene expression, rutin biosynthesis

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INTRODUCTION Fagopyrum esculentum Moench is the main cultivated species of the genus Fagopyrum. F. esculentum, common buckwheat, originates from Southwest China and has gradually spread around the world. The flavonoid content and composition in buckwheat seeds differs among species. F. esculentum and F. tataricum (Tartary buckwheat, another cultivated species) have been well studied and compared in previous research. Both common and Tartary buckwheat contain high levels of nutrients and phenolics, amino acids,1 minerals, and dietary fiber.2 It has been reported that rutin, quercetin, and quercitin, the main flavonoids in buckwheat, are higher in Tartary than in common buckwheat.3,4 Other active compounds, including emodin,5 hypericin, fagopyrin,6 and taroside,7 have also been reported from buckwheat. Buckwheat is considered an important crop with both agricultural and pharmaceutical value. Beneficial properties that have been reported in buckwheat include antioxidant activity,8−10 antitumor activity,11 and hypolipidemic effects.12 It has recently been reported that consumption of buckwheat modulates the postprandial response of selected gastrointestinal satiety hormones in individuals with type 2 diabetes mellitus.13 Rutin and quercetin from Tartary buckwheat were demonstrated to prevent liver inflammatory injury by promoting antioxidative and anti-inflammatory activity against oxidative liver damage.14 © 2014 American Chemical Society

Genetic and environmental factors can affect the nutritional content of buckwheat.1,3,15,16 It has been reported that salicylic acid treatment (150 mg/L) enhanced rutin content in F. tataricum and upregulated the expression of rutin biosynthesisrelated genes.17 L-Phentermine and UV-C treatment have been reported to increase rutin content in buckwheat seedlings.18 Common buckwheat is a self-incompatible species with outcrossing characteristics. Various plant-breeding trials have been conducted in an attempt to obtain favorable characteristics or increase nutritional value by optimizing rutin content in buckwheat.16,19 In the present study, we detected transcription patterns and expression levels of a set of key enzymes belonging to the flavonoid biosynthetic pathway by quantitative real-time polymerase chain reaction analysis and examined the production of flavonoids and free amino acids in two common F. esculentum cultivars. Hydrophilic metabolic profiling in common buckwheat using gas chromatography−time-of-flight mass spectrometry (GC-TOFMS) and principal component analysis (PCA) were applied to determine phenotypic variation between the cultivars. Finally, we compared and discussed the nutritional value of two common F. esculentum cultivars. Received: Revised: Accepted: Published: 2701

November 4, 2013 February 7, 2014 March 3, 2014 March 3, 2014 dx.doi.org/10.1021/jf4049534 | J. Agric. Food Chem. 2014, 62, 2701−2708

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Figure 1. Expression levels of flavonoid biosynthesis genes in two different cultivars of Fagopyrum esculentum. T-Ori indicates Tanno-original common buckwheat; T-R indicates Tanno-rutin cultivar. The expression level of each gene is relative to that of the constitutively expressed histone H3 gene. Each value is the mean of three replicates ± SD.

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housekeeping gene.21 QRT-PCR reactions were performed in triplicate on a MiniOpticon system (Bio-Rad Laboratories, Hercules, CA) with the Quantitect SYBR Green PCR Kit (Qiagen, Valencia, CA). The PCR protocol was as follows: denaturation for 5 min at 95 °C followed by 40 cycles of denaturation for 15 s at 95 °C, annealing for 15 s at 56 °C, and elongation for 20 s at 72 °C. PCR results were calculated as the mean of three replicated treatments. Extraction of Flavonoids in Buckwheat. Plant materials were freeze-dried and then ground to a powder before extraction using methods described previously with some modification.22 Powdered samples (0.10 g) were extracted with 5 mL of 80% methanol (MeOH) containing 10% acetic acid (0.1% v/v); this was followed by vortexing for 5 min at room temperature, and then the samples were extracted for 1 h at 37 °C, with 1 min of vortex every 20 min. After centrifugation at 1000 × g for 5 min, the supernatant was filtered and used for analysis. Quantitative Analysis of Phytochemicals by HPLC. External standards were purchased from Extrasynthese (Genay, France). HPLC-grade methanol (MeOH) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Acetic acid was provided from

MATERIALS AND METHODS

Plant Materials and Culture Conditions. Two common buckwheat cultivars, ‘Tanno-Hiushinai original line’ (Tanno-original) and ‘Tanno-Hiushinai high rutin line’ (Tanno-rutin) were bred by the Hokkaido Agricultural Research Center (Hokkaido, Japan). Seeds of the two buckwheat cultivars were germinated and cultured in a greenhouse (25 °C, 50% humidity) at Chungnam National University (Daejeon, Korea). The various plant organs were harvested when seeds were mature. Samples were rapidly frozen in liquid nitrogen after collection and stored at −80 °C until analysis. Total RNA Extraction and Quantification of Gene Expression. Total RNA was isolated from samples of F. esculentum using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA). For quantitative realtime polymerase chain reaction (qRT-PCR), 1 μg of total RNA was reverse transcribed using the Superscript II First-Strand Synthesis Kit (Invitrogen, Carlsbad, CA) and an oligo(dT)20 primer. Transcription levels were analyzed by qRT-PCR. The gene-specific primer sets were designed for qRT-PCR as described previously.20 Gene expression was normalized to that of the histone H3 gene as a 2702

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Jun Sei Chemical Co., Ltd. (Kyoto, Japan). For HPLC analysis, extracts were filtered through a 0.45-μm poly filter and diluted 2-fold with MeOH prior to HPLC analysis. Samples were determined using a Futecs model NS-4000 HPLC apparatus (Daejeon, Korea) with a UV−vis detector and autosampler. The analysis was monitored at 280 nm and performed using a C18 column (250 mm × 4.6 mm, 5 μm; RStech, Daejeon, Korea). The mobile phase consisted of a mixture of (A) MeOH/water/acetic acid (5:92.5:2.5, v/v/v) and (B) MeOH/ water/acetic acid (95:2.5:2.5, v/v/v); the procedure was described previously.23 The initial mobile phase composition was 0% solvent B, followed by a linear gradient from 0% to 80% of solvent B in 48 min, then holding at 0% solvent B for an additional 10 min, and the column was maintained at 30 °C. The flow rate was set at 1.0 mL/min, and the injection volume was 20 μL. Quantification of the different compounds was based on peak areas and calculated as equivalents of representative standard compounds. All contents were expressed as microgram per milligram dry weight. GC-TOFMS Analysis of Polar Metabolites. Polar metabolite extraction was performed as described previously.24 A total of 20 mg of ground sample was extracted with 1 mL of a mixed solvent of methanol/water/chloroform (2.5:1:1, v/v/v). Ribitol solution (120 μL, 0.2 mg/mL) was added as an internal standard (IS). Extraction was performed at 37 °C with a mixing frequency of 1200 rpm for 30 min, using a thermomixer compact (Eppendorf AG, Germany). The solutions were then centrifuged at 16000 × g for 3 min. The polar phase (0.8 mL) was transferred into a new tube, and 0.4 mL of water was added before centrifugation to separate the nonpolar phase. The mixed contents of the tube were centrifuged at 16000 × g for 3 min. The methanol/water phase containing hydrophilic metabolites was dried in a centrifugal concentrator (CVE-2000, Eyela, Japan) for 2 h, followed by drying in a freeze-dryer for 16 h. Methoxime (MO) derivatization was performed by addition of 160 μL of methoxyamine hydrochloride (20 mg/mL) in pyridine and shaking at 30 °C for 90 min. Trimethylsilyl (TMS) etherification was performed by addition of 160 μL of MSTFA at 37 °C for 30 min. GC-TOFMS was performed using an Agilent 7890A gas chromatograph (Agilent, Atlanta, GA, USA) coupled to a Pegasus HT TOF mass spectrometer (LECO, St. Joseph, MI). Derivatized sample (1 μL) was separated on a fused-silica capillary column (30 m × 0.25 mm id) coated with 0.25 mm of CPSIL 8 CB low bleed (Varian Inc., Palo Alto, CA, USA). The split ratio was set at 1:25. The injector temperature was 230 °C, and the flow rate of helium through the column was 1.0 mL/min. The temperature program was as follows: initial temperature of 80 °C for 2 min, followed by an increase to 320 °C at 15 °C/min, and a 10 min hold at 320 °C. The transfer line and ion-source temperatures were 250 and 200 °C, respectively. The scanned mass range was 85−600 m/z, and the detector voltage was set at 1700 V. The quantification of each analyte was based on the peak area ratio relative to that of the IS. Statistical Analysis. Each result shown in the figure or table was the mean of three replicated treatments. Data were expressed as the mean of three independent replicates. The significant differences between treatments were statistically evaluated by the statistical analysis software (SAS), version 8.2. The GC-TOFMS data were subjected to principal component analysis (PCA) (SIMCA-P, version 13.0, Umetrics, Umeå, Sweden) to evaluate similarity/dissimilarity among groups of multivariate data. The PCA output consisted of score plots to visualize the contrast between samples and loading plots to explain the cluster separation. Unit variance scaling was used without transformation of the data.

higher transcription levels in stems of the high-rutin cultivar compared with the original cultivar. In particular, the PAL gene, which plays a primary role in the flavonoid biosynthetic pathway, showed 3- and 4-fold higher expression in stems and leaves of the high-rutin cultivar. Compared with the original cultivar, FeC4H, FeF3H, and FeDFR also maintained higher transcript level in leaves of the high-rutin cultivar. However, the transcription levels of FePAL, Fe4CL1, FeC4H, FeCHS, FeF3H, FeF3′H, FeFLS1, and FeDFR were higher in roots of the original cultivar. Transcription of isoforms or multicopies of CHS, CHI, 4CL, F3H, and FLS has shown the different expression levels on the regulation of the flavonoid biosynthetic pathway in many plants.25−29 4CL is involved in lignification and flavonoid biosynthesis; 4CL ligates coenzyme A (CoA) with hydroxycinnamic acids, such as 4-coumaric acid and caffeic acid to form the corresponding CoA thioesters.30,31 In the present study, two 4CL isoform genes showed different transcriptional levels in buckwheat organs, and both genes exhibited higher expression in the roots and stems of buckwheat than in the leaves and seeds. This result is consistent with the physiological and morphological structures of the buckwheat plant. Fe4CL2 showed higher activity than Fe4CL1 in the two buckwheat cultivars (Figure 2); in particular, the highest level of Fe4CL2

Figure 2. Expression levels of flavonoid biosynthesis genes in seeds of two different cultivars of Fagopyrum esculentum. T-Ori indicates Tanno-original common buckwheat; T-R indicates Tanno-rutin cultivar. The expression level of each gene is relative to that of the constitutively expressed histone H3 gene. Each value is the mean of three replicates ± SD.

was observed in the stems of the high-rutin cultivar. Overall, our results indicated that the two 4CL isoforms may have the different functions in lignification and flavonoid biosynthesis in buckwheat. FLS is an important enzyme in the synthesis of quercetin, which is the precursor of rutin.32 Two FLS isoform genes have been reported in tartary buckwheat, which showed differential expression and regulation levels in different buckwheat cultivars and under stress conditions.32 In the present study, the two FLS isoform genes exhibited differential transcriptional levels in the original and high-rutin common buckwheat cultivars. Our results indicated that expression of FeFLS2 was obviously higher in seeds of the high-rutin cultivar than seeds of the original cultivar (Figure 2). The expression level of FeFLS1 in seeds was not significantly different between the cultivars. However, expression of FeFLS2 in roots, stems, and leaves was 2−4 times higher in the original than in the high-rutin cultivar.

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RESULTS AND DISCUSSION Expression of Flavonoid Biosynthetic Genes in Buckwheat. The flavonoid biosynthetic pathway is well described in plants. In this study, expression of ten flavonoid biosynthetic pathway genes (FePAL, FeC4H, Fe4CL, FeCHS, FeCHI, FeF3H, FeF3′H, FeFLS, FeDFR, and FeANS) was detected in the two cultivars of common buckwheat by qRTPCR (Figure 1). Most genes, FePAL, Fe4CL1, Fe4CL2, FeC4H, FeCHS, FeCHI, FeF3H, FeF3′H, FeDFR, and FeANS, showed 2703

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Figure 3. The accumulation of phytochemicals in two Fagopyrum esculentum cultivars. The plotted data and error bars indicate the means and variance of duplicate individual experiments. Each value is the mean of three replicates ± SD.

Phytochemical Content in Buckwheat. The compounds gallic acid, 4-hydroxybenzoic acid, caffeic acid, benzoic acid, rutin, trans-cinnamic acid, quercetin, and kaempferol were detected by HPLC analysis (Figure 3). Gallic acid, 4hydroxybenzoic acid, caffeic acid, benzoic acid, and rutin were highly detected in leaves and stems of the high-rutin cultivar. trans-Cinnamic acid was 6-fold higher in roots of the original cultivar, and kaempferol was higher in roots than in other organs, particularly in the original cultivar. The primary active component, rutin, showed the highest levels of accumulation in leaves, followed by stems and seeds, and roots contained the lowest levels of this compound. Rutin content in leaves, stems, and seeds of the high-rutin cultivar were 24.86, 1.46, and 1.36 μg/mg, respectively, which was 1.73-, 1.62-, and 1.77-fold higher than that in the original buckwheat cultivar. Buckwheat is a beneficial and healthy source of dietary rutin, and it has excellent rutin levels compared with those in other crops.33−36 Rutin content in buckwheat depends on the variety and environmental factors.37−40 Rutin content has shown significant variations in different growth stages of different Fagopyrum species.37 The results of our study also proved that the quality of buckwheat, especially rutin content could be improved by breeding method. Our results indicated that the higher rutin content in high-rutin cultivar seeds may be directly related to FtFLS2, which showed higher expression levels in

high-rutin cultivar seeds (Figure 2). However, even the expression pattern of PAL is correlated with its direct product, cinnamic acid, in the roots of buckwheat; this trend was not observed in the stems and leaves of the two buckwheat cultivars. The relationship between rutin content and gene expression of PAL and CHS did not show any obvious positive or negative correlation, this result was not consistent with the finding of a previous study.37 Our results showed that the levels of flavonoid biosynthetic genes did not clearly correlate with rutin and other flavonoids in buckwheat. Similar results have been reported by previous studies,32,41−43 which leads us to speculate that these results may be attributable to the dynamic transport of metabolites during physiological and morphological processes in plants. Otherwise, the rutin-degrading enzyme would be another factor that influences the rutin levels in different organs of buckwheat.44,45 In addition, secondary metabolism in plants is complicated; it could be co-regulated by a complex set of regulatory networks, that is, by not only structural genes but also other classes of genes, including regulators, modifiers, transporters, and elicitors.38,39,46−48 cDNA amplified fragment length polymorphism (AFLP) has shown that several factors involved in the regulation, metabolism, signaling, and transport of secondary metabolites contribute to the higher rutin content and overall nutritional superiority of rice-tartary over common buckwheat,48 Study of 2704

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Figure 4. Scores (A) and loading plots (B) of principal components 1 and 2 of the PCA results from polar metabolite data for Tanno-rutin and Tanno-original buckwheat cultivars. T-Ori indicates Tanno-original common buckwheat; T-R indicates Tanno-rutin cultivar.

the differences in rutin or other nutritional content between Tanno-original and Tanno-rutin cultivars may be required. Metabolic Profiling of Buckwheat. The production of secondary metabolites is strongly associated with primary

metabolism pathways. Thus, we conducted comprehensive metabolic phenotyping of the primary metabolism in the peels and flower organs of buckwheat. GC-TOFMS has been one of the most popular metabolomic techniques because it can 2705

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basis for the high rutin content in a common buckwheat cultivar by employing the metabolomic method of analysis.

determine the levels of primary metabolites such as amino acids, organic acids, and sugars by using chemical derivatization of these hydrophilic metabolites. In this study, low-molecularweight molecules from peels and flower organs were identified using GC-TOFMS. The advantages of GC-TOFMS are the relatively high reproducibility and possible adaption for highthroughput analysis because of its rapid spectrum accumulation times. Furthermore, use of mathematical algorithms for deconvolution of closely overlapping peaks is possible because of the high mass-spectrum similarities among peaks. Forty-six metabolites, including 17 amino acids, 17 organic acids, 8 sugars, 3 sugar alcohols, and 1 amine, were detected in the peels and seed flour of two cultivars of F. esculentum by using GCTOFMS (Supplementary Figure S1 and Table S1, Supporting Information). Quantification was performed using selected ions (Supplementary Table S1, Supporting Information). According to the PCA analysis (Figure 4), the two highestranking principal components (PCs) accounted for 87.3% of the total variance within the data set. The first PC (PC1), accounting for 70.1% of total variance, resolved the measured metabolite profiles of buckwheat peels and flour. The second PC (PC2, 17.5% of total variance) differentiated the high-rutin from the original cultivar. There was a clear absence of significant variance within tissues of the same cultivar. PCA enables identification of compounds that exhibit the greatest variance within a population and allows closely related compounds to be identified. In PC2, the variation was mainly attributable to galactose and glucose, for which the eigenvectors were 0.34217 and 0.32803, respectively. The building blocks for secondary metabolites are derived from primary metabolism. Rutin (also called quercetin-3-O-rutinoside and sophorin) is the glycoside between the flavonol quercetin and the disaccharide rutinose. Thus, the levels of endogenous glucose may be lower in the high-rutin cultivar than in the low-rutin cultivar because of glycosylation reactions.49,50 Our results suggest that these metabolites were present in lower quantities in the Tanno-rutin buckwheat than in Tanno-original. Our results showed that buckwheat peels contained amino acids and carbohydrates similar to those in flour. Tanno-original buckwheat contained a particularly high level of carbohydrates (fructose, glucose, mannose, mannitol, and sucrose) in both peels and flour (Supplementary Table S2, Supporting Information). In this study, metabolites and flavonoid biosynthetic pathway-related genes were compared between Tanno-original and Tanno-rutin, two cultivars of common buckwheat. We found that the Tanno-rutin cultivar generally had higher rutin content in the aerial plant, indicating it could be a good alternative to culture for agricultural usage. Previously, it has been reported that hulls and flour of common buckwheat contained phenolics, flavonols, and procyanidins, and that flavonoids were more abundant in hulls than in flour.51 The bran is considered a byproduct of buckwheat and has been proven to be a good source of flavonoids with antioxidant and antibacterial activities.51,52 In this study, we reported the metabolites in peels and flours of the two buckwheat cultivars, which provided nutritional information about the seeds of these cultivars. The hydrophilic metabolite profile is closely related to an organism’s phenotype and reveals important nutritional characteristics.53,54 The differences between these two buckwheat cultivar seeds were identified by PCA results from the metabolite data. Even Tartary buckwheat has a high content of rutin,3,4 but common buckwheat is cultivated more widely worldwide. This is the first report to present the theoretical

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ASSOCIATED CONTENT

S Supporting Information *

Metabolites identified in peels and seed flour of the two cultivars and results of PCA. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*S. U. Park. Phone: +82-42-821-5730. Fax: +82-42-822-2631. E-mail: [email protected]. Author Contributions §

Xiaohua Li and Jae Kwang Kim contributed equally to this work. Funding

This work (K11101) was supported by the Korea Institute of Oriental Medicine (KIOM) grant funded by the Korea government and also partially carried with the support of "Next-Generation BioGreen 21 Program (SSAC, Project No. 009520)" Rural Development Administration, Republic of Korea. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr.Tastsuro Suzuki (Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization, Japan) for providing two common buckwheat cultivars, ‘TannoHiushinai original line’ and ‘Tanno-Hiushinai high rutin line’, used in this work.

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ABBREVIATIONS USED HPLC, high-performance liquid chromatography; PAL, phenylalanine ammonium lyase; C4H, cinnamic acid 4-hydroxylase; CHI, chalcone isomerase; CHS, chalcone synthase; 4CL, 4coumarate-CoA ligase; DFR, dihydroflavonol reductase; F3H, flavanone-3-hydroxylase; F3′H, flavonoid-3′-hydroxylase; FLS, flavonol synthase; ANS, anthocyanin synthase

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