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Article

An Evaluation of the Absolute Content of Flavonoids and the Identification of Their Relationship with the Flavonoid Biosynthesis Genes in Tartary Buckwheat Seeds

1
School of Life Sciences, Guizhou Normal University, Guiyang 550025, China
2
Research Center of Buckwheat Industry Technology, Guizhou Normal University, Guiyang 550001, China
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Agronomy 2023, 13(12), 3006; https://doi.org/10.3390/agronomy13123006
Submission received: 15 October 2023 / Revised: 20 November 2023 / Accepted: 27 November 2023 / Published: 7 December 2023
(This article belongs to the Special Issue Seeds: Chips of Agriculture)

Abstract

:
The aim of the present study is to evaluate the absolute content and accumulation patterns of flavonoid components; to give insight into the accumulation relationships among flavonoid components; to explore the correlation between the content of flavonoid components and the expression of flavonoid biosynthesis genes in Tartary buckwheat seeds; and to construct a biosynthetic pathway on the major flavonoid components in Tartary buckwheat seeds. In total, 61 flavonoid components were absolutely quantified in five Tartary buckwheat varieties, of which 41 existed in all varieties. The content of most flavonoids varied significantly among different varieties or within the same variety. Rutin, quercetin, nicotiflorin, and kaempferol were the dominant flavonoid components in the Tartary buckwheat seeds, accounting for 73.05–81.79% of the total flavonoids. Significantly positive or negative correlations with content accumulation were found between some flavonoid components. Thirty-six flavonoid components displayed four different accumulation patterns in the developing Tartary buckwheat seeds. Seventeen structural genes for flavonoid biosynthesis displayed a significantly positive correlation with the accumulation of most flavonoid components during the development of Tartary buckwheat seeds, and the F3′5′H-3 gene might be the most crucial contributor in determining the total flavonoid content in Tartary buckwheat seeds. A schematic of the biosynthesis pathways for 30 major flavonoids in Tartary buckwheat seeds was constructed. These findings provide an outlook of the flavonoid components and their biosynthesis in Tartary buckwheat seeds and have potential applications in breeding new cultivars with higher flavonoid contents.

1. Introduction

Flavonoids are the most abundant secondary metabolites in plants, and are synthesized from phenylalanine. To date, approximately 10,000 flavonoids have been identified in plants [1]. They not only contribute to a plant’s coloration, development, and biotic and abiotic stress resistance but also play crucial roles in human health through their high antioxidant capacities, including anti-cancer, antitumor, anti-hypertension, anti-inflammatory, anti-diabetic, vascular protection, neuro-protection, cardio-protection, and hepatoprotection [2,3,4,5,6,7,8].
Tartary buckwheat (Fagopyrum tataricum Gaertn.), originating from southwestern China, is an important pseudocereal crop and is broadly cultivated in Asia and Eastern Europe [9,10].
In recent years, Tartary buckwheat has been highly valued due to its significant health care and pharmaceutical effects on the human body, and its high content of bioactive flavonoids is the main contributor to its high health care value. A lot of studies have demonstrated that flavonoids in Tartary buckwheat have positive effects on hypertension, diabetes, liver injury, vascular disease, oxidative stress, cancer, and so on [9,11,12,13,14,15].
The seed is the main utilized part of Tartary buckwheat. It contains a high content of bioactive flavonoids (0.67–2.27%) and abundant flavonoid components and is especially rich in rutin (accounting for 70–85% of the total flavonoids), which is not found in other major crops (rice, wheat, and maize) [16,17]. Therefore, the seeds of Tartary buckwheat have been widely used in creating various health products, including tea, noodles, cookies, wine, and so on. In addition, in the study of the flavonoid components in Tartary buckwheat seeds, 93 flavonoid components have been determined by using ultra-high-performance liquid chromatography coupled with the triple quadrupole mass spectrometry (UHPLC-QqQ-MS) metabolomics approach [18]. Of these, 32 flavonoid components were predicted to play crucial roles in the resistance of cancers/tumors, atherosclerosis, hypertension, diabetes, cardiovascular disease, atherosclerosis, and thrombotic disease [18]. However, apart from rutin, quercetin, kaempferol, and nicotiflorin, the absolute content of other flavonoid components in Tartary buckwheat seeds is largely unclear [19,20]. Furthermore, the entire life cycle accumulation patterns have only been investigated for rutin, quercetin, and kaempferol, and other flavonoid components still remain to be explored [21]. More importantly, the inter-relationships between flavonoid components in Tartary buckwheat seeds are also unclear. Similarly, in respect of flavonoid biosynthesis genes, although the expression patterns of some flavonoid biosynthesis genes and the correlation between their expression and the accumulation of some flavonoid components have been investigated in developing Tartary buckwheat seeds, these studies only focus on three stages rather than the entire life cycle of the seeds [9,21]. This limits the understanding of flavonoid components in Tartary buckwheat seeds and the breeding of new varieties with high contents of specific flavonoid components.
Therefore, the object of this work is to determine the absolute content of different flavonoid components in Tartary buckwheat seeds; to investigate the accumulation patterns of flavonoid components in the seeds across the entire life cycle; to elucidate the correlations among flavonoid components; and to reveal the relationship between their accumulation and the expression of flavonoid biosynthesis genes. This study provides valuable information for the breeding of new varieties with high contents of total flavonoids or specific flavonoid components.

2. Materials and Methods

2.1. Plant Materials

Five Tartary buckwheat varieties (Pinshan, TB01; Heiku2, TB02; Heiku 10; TB03; Shaobai, TB04; Kuqiao298, TB05), which originated from the Research Center of Buckwheat Industry Technology of Guizhou Normal University (Guiyang, Guizhou, China), were used in this study. These varieties were planted in a field in Anshun, Guizhou province, China (Lat. 26°19′ N, 105°59′ E, Alt. 1433 m), in August 2022. The same field management was performed for all varieties during plant growth. At maturity (December 2022), the seeds of these five varieties were collected and used to determine the total content and the absolute content of flavonoid components. Furthermore, the flowers of the TB02 variety that had only just fully opened (finished pollination) were tagged, and the seeds were collected at the filling (10, 13, and 16 DAP (day after pollination)) and maturing (20, 25, and 30 DAP) stages. All of these sample collections were performed on three biological replicates. The collected seeds in different developing stages were immediately frozen with liquid nitrogen and then stored at −80 °C.

2.2. Determination of Total Flavonoid

The content of total flavonoids was quantified using a previously described aluminum chloride colorimetric method [20]. In brief, the shelled seeds were ground into a fine powder. Then, 0.1 g of this powder was used to extract the total flavonoids via incubation in 70% methanol at 65 °C with oscillated shaking. After centrifugation, 70% methanol was added to the fluid extracts for a total volume of 5 mL and left to stand for 30 min. Then, the absorbance of the mixture at 420 nm was measured using a microplate reader (Thermo, Franklin, MA, USA). The total flavonoid content was calculated using the rutin calibration curve (R2 = 0.999).

2.3. Determination of the Absolute Content of Flavonoid Components

The qualitative and absolute quantitative determination of flavonoid components in the five Tartary buckwheat seeds were performed at Metware Biotechnology Co., Ltd. (Wuhan, China), where qualitative and absolute quantitative analyses of 204 flavonoid components can be conducted simultaneously (Table S1). The experimental methods were also provided by this company.

2.3.1. Sample Preparation and Extraction

The seeds were ground into a powder using a ball crusher (30 Hz, 1.5 min). Then, 20 mg of this powder was used to extract the total flavonoids with 0.5 mL of 70% methanol, and 10 μL of an internal standard (IS; 4000 nmol/L) was added to the extract for the quantitation of flavonoid components (Table S1). The extract was sonicated for 30 min and centrifuged at 12,000× g under 4 °C for 5 min. The supernatant was filtered through a 0.22 μm membrane filter for further LC-MS/MS analysis. For the analysis of rutin, quercetin, and nicotiflorin, the sample extracts were diluted 1000 times.

2.3.2. UPLC Conditions

The sample extracts were analyzed using an UPLC-ESI-MS/MS system (UPLC, ExionLC™ AD, https://sciex.com.cn/ (accessed on 13 February 2023); MS, Applied Biosystems 6500 (Framingham, MA, USA) Triple Quadrupole, https://sciex.com.cn/). The analytical conditions were as follows: UPLC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 μm, 2.1 mm × 100 mm); solvent system, water with 0.05% formic acid (A), acetonitrile with 0.05% formic acid (B). The gradient elution program was set as follows: 0–1 min, 10–20% B; 1–9 min, 20–70% B; 9–12.5 min, 70–95% B; 12.5–13.5 min, 95% B; 13.5–13.6 min, 95–10% B; and 13.6–15 min, 10% B. The flow rate was set at 0.35 mL/min and the temperature was set at 40 °C. The injection volume was 2 μL.

2.3.3. ESI-MS/MS Conditions

Linear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on a triple quadrupole linear ion trap mass spectrometer (QTRAP), QTRAP® 6500+ LC-MS/MS System (Framingham, MA, USA), equipped with an ESI Turbo Ion-Spray interface, operating in the positive and negative ion modes and controlled by Analyst 1.6.3 software (AB Sciex, Warrington, UK). The ESI source operation parameters were as follows: ion source, ESI+/−; source temperature, 550 °C; ion spray (IS) voltages, 5500 V (positive) and −4500 V (negative); and curtain gas (CUR) was set at 35 psi. The flavonoids were analyzed using scheduled multiple reaction monitoring (MRM). Data were acquired using Analyst 1.6.3 software (Sciex). Multiquant 3.0.3 software (Sciex) was used to quantify all metabolites. The mass spectrometer parameters, including the declustering potentials (DPs) and collision energies (CEs) for individual MRM transitions, were further DP and CE optimized. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period [22].

2.3.4. Analysis of the Absolute Content of Flavonoid Components

The standard solution for 204 flavonoid components was prepared at different concentrations (0.5 nmol/L, 1 nmol/L, 5 nmol/L, 10 nmol/L, 20 nmol/L, 50 nmol/L, 100 nmol/L, 200 nmol/L, 500 nmol/L, 1000 nmol/L, and 2000 nmol/L), and the calibration curve was constructed with concentration as the abscissa and with peak area as the ordinate. Then, the absolute content of each of the detected flavonoid components was obtained by substituting its peak area into the standard curve linear equation for calculation.

2.4. Investigation of the Accumulation Pattern of Flavonoid Component during Seed Development

To investigate the accumulation pattern of flavonoid components in developing seeds of Tartary buckwheat, a widely targeted metabolomics analysis was performed for TB02 seeds at six different developing stages (10, 13, 16, 20, 25, and 30 DAP). The relative content for these abovementioned detected flavonoid components was obtained in each sample, and the heatmaps were constructed with TBtools 2.012 [23].

2.5. Construction of Flavonoid Biosynthesis Pathway in Tartary Buckwheat Seeds

Forty-one flavonoid components absolutely quantified in the seeds of all five Tartary buckwheat varieties were used to construct the predicted flavonoid biosynthesis pathway in Tartary buckwheat seeds. The reported flavonoid biosynthesis pathways in buckwheat can be used as references [24,25].

2.6. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis of the Structural Genes for Flavonoid Biosynthesis in the Seeds of Different Tartary Buckwheat Varieties

Six structural genes for flavonoid biosynthesis (FtCHI, FtF3H, FtF3′H, FtF3′5′H, FtFLS, and FtUGT79A15) were selected for qRT-PCR with three biological replicates. Total RNA was isolated from the seeds of five Tartary buckwheat varieties at 13 DAP using the EASYspin Plus Plant RNA Kit. The reverse transcription and qRT-PCR reactions were carried out according to a previously described method [9]. The primers used in qRT-PCR are listed in Table S2.

2.7. Investigation of the Expression Pattern of the Structural Genes for Flavonoid Biosynthesis

The expression patterns of 17 flavonoid biosynthesis structural genes, including 4 CHS, 1 CHI, 2 F3H, 1 F3′H, 1 FLS, 3 FtF3′5′H, 1 DFR, 1 ANR,1 ANS, 1 LAR, and 1 UGT (FtUGT79A15), were investigated in the developing seeds (10, 13, 16, 20, 25, and 30 DAP) using our transcriptome data (unpublished data). TBtools was used to construct the heat map of gene expression [23].

2.8. Identification of the UDP-Glycosyltransferase (UGTs) Genes Involved in the Biosynthesis of Flavonoid Glycosides in Tartary Buckwheat Seeds

To identify the UDP-glycosyltransferase genes that participated in the biosynthesis of flavonoid glycosides in Tartary buckwheat seeds, 42 UGT genes, which were identified by Li et al. [9] as having different expressions during in Tartary buckwheat seeds, were selected. The expression values of the 42 UGT genes were obtained from the seed transcriptome data of TB02 at six developing stages. The heat map of 42 UGT genes’ expressions and 16 flavonoid glycosides’ contents was constructed using TBtools [23]. The correlation among the expression value of 42 UGT genes and the content of 16 flavonoid glycosides were analyzed during seed development, and UGT genes with r > 0.7 and a p-value of <0.05 were considered candidate genes that related to the biosynthesis of flavonoid glycosides in Tartary buckwheat seeds.

2.9. Statistical Analysis

For each sample, the mean value was obtained from three replicates. A one-way analysis of variance (ANOVA) was performed, and a p-value of <0.05 was defined as statistically significant. The content correlation among flavonoid components was calculated using Microsoft Excel (Redmond, WA, USA).

3. Results and Discussion

3.1. Total Flavonoid Content in the Seeds of Tartary Buckwheat Varieties

The total flavonoid content in the seeds of five Tartary buckwheat varieties was determined, ranging from 1.67% (TB05) to 1.89% (TB01), with a mean of 1.78% (Figure 1). Furthermore, there was a significant difference in the total flavonoid content among TB01, TB03, TB04, and TB05. The results were similar to those in a previous report where the total flavonoid content in the seeds of different Tartary buckwheat varieties varied significantly and ranged from 0.61% to 3.04% [10,16,25,26].

3.2. Composition and Absolute Content of Flavonoid Components in Seeds of Different Tartary Buckwheat Varieties

In Tartary buckwheat, a total of 93 flavonoids have been identified in seeds using UHPLC-QqQ-MS analysis [18]. However, most of their absolute contents remain unclear. Here, we obtained the absolute content of 61 flavonoids from five different Tartary buckwheat varieties (Figure 2), displayed in Table 1. A total of 45, 51, 48, 44, and 52 flavonoids were quantified in TB01, TB02, TB03, TB04, and TB05, respectively. Among these 61 flavonoids, 41 were detected in all five varieties, and 2, 4, 1, and 5 flavonoids were specifically detected in TB01, TB02, TB03, and TB05, respectively (Figure 2). Furthermore, the content of most flavonoids among different varieties or within the same variety were significantly different (Table 1). Among these 61 flavonoids, only 11 (rutin, quercetin, nicotiflorin, kaempferol, quercimeritrin, hesperidin, procyanidin B2, epicatechin, narcissin, catechin, and astragalin) had a content of >1 μg/g in all five Tartary buckwheat varieties, and the remaining of 50 had very low contents (<1 μg/g). The four flavonoids with the highest content were rutin (9021.432–12,434.997 μg/g), quercetin (1083.64–2290.068 μg/g), nicotiflorin (543.077–1016.949 μg/g), and kaempferol (75.353–142.295 μg/g) in all five varieties, which accounted for 73.05–81.79% of the total flavonoids (Table 1). These results are in agreement with those of previous reports, in which it was found that rutin, quercetin, and nicotiflorin are the major flavonoid components in Tartary buckwheat seeds [19,26,27,28]. Tartary buckwheat has been found to have a wide variety of benefits for human health due to its higher content of bioactive flavonoids [11,12,13,14,15,29,30,31]. Therefore, among the flavonoids of buckwheat seeds, rutin, quercetin, and nicotiflorin are considered the major contributors beneficial to human health. It has been reported that isoflavones, which have only been identified in some leguminous plants, display female hormone-like and cancer- prevention activities, in addition to having the same function as other flavonoids [9,32,33]. In the developing seeds with shell of Tartary buckwheat, ten isoflavones have been identified using metabolomics technology [9]. However, their absolute content remains unclear. In our results, one isoflavone (glycitin) was determined in TB03 (0.144 ± 0.012 μg/g), suggesting that isoflavone is present in Tartary buckwheat seeds but that this presence might be variety-specific. Therefore, besides leguminous plants, Tartary buckwheat varieties containing isoflavones can be considered a new source of isoflavones for humans.

3.3. Correlation among Flavonoid Components and Expression of Structural Genes for Flavonoid Biosynthesis in Tartary Buckwheat Seeds

To explore the correlations among the accumulated flavonoid, which is currently unclear, we analyzed the correlation coefficients of 20 flavonoids with the highest content in Tartary buckwheat seeds. As shown in Figure 3, each of the twenty flavonoids displayed a significantly positive (r > 0.6, p < 0.05) or negative (r < −0.6, p < 0.05, except taxifolin and naringenin-7-glucoside) correlation with at least one flavonoid component (Table S3). Among the 20 flavonoids, avicularin, and kaempferol displayed the most positive and negative correlations with other flavonoids, respectively. Notably, nicotiflorin (kaempferol-3-O-rutinoside) displayed a significantly positive correlation with kaempferol (r = 0.8, p = 0.000032) but significantly negative correlations with baimaside (quercetin 3-O-sophoroside) (r = −0.62, p = 0.000009) and spiraeoside (quercetin-4-O-glucoside) (r = −0.72, p = 0.000009) (Figure 3 and Table S3). Considering that kaempferol was the substrate for the biosynthesis of nicotiflorin and quercetin, quercetin was the substrate for the biosynthesis of baimaside and spiraeoside, and dihydrokaempferol was precursor for the biosynthesis of both kaempferol and quercetin, our results indicated that the correlation among flavonoid components in Tartary buckwheat seeds was reliable and that there is competition in the biosynthesis of flavonoids with the same substrate source. Among the four flavonoids with the highest contents, rutin showed significantly negative correlations with quercetin (r = −0.97, p = 0.000000327) and kaempferol (r = −0.76, p = 0.000000074) and quercetin displayed a significantly negative correlation with kaempferol (r = 0.8, p = 0.000126) (Table S3). All of the above results implied that it was difficult to simultaneously increase the content of these four major flavonoids in Tartary buckwheat seeds through breeding improvements. In addition, all of the correlation results suggest that flavonoid biosynthesis is very complex and involves different metabolic fluxes in Tartary buckwheat seeds.
To further investigate whether the different expressions of the structural genes for flavonoid biosynthesis caused differences in the content of flavonoid components in different Tartary buckwheat variety seeds, we performed a qRT-PCR analysis of six structural genes for flavonoid biosynthesis in the seeds of five Tartary buckwheat varieties at 13 DAP, which was a crucial period of flavonoid accumulation during seed development [9]. As a result, there were significant differences in the expression of FtF3′H, FtF3′5′H, and FtUGT79A15 among the five Tartary buckwheat varieties (Figure 4). Notably, the encoding protein of FtUGT79A15 was demonstrated to be able to catalyze the quercetin 3-O-glucoside synthesis of rutin in vitro and in planta [34]. Consistent with the expression of FtUGT79A15, the rutin content was significantly different among different Tartary buckwheat varieties. This suggests that the different expressions of FtUGT79A15 in the seeds might be the reason for the difference in the accumulation of rutin. In addition, the differential expression of FtF3′H and FtF3′5′H might also have determined the different contents of the flavonoid components in the different Tartary buckwheat variety seeds.

3.4. Accumulation Patterns of Flavonoid Components and Expression Patterns of Structural Genes for Flavonoid Biosynthesis in the Developing Tartary Buckwheat Seeds

To date, the accumulation trends of some flavonoid components have been investigated in different tissues [9,35]. However, the accumulation pattern of flavonoids in seeds across its entire life cycle remained unclear. Here, 41 flavonoid components, which existed in all five determined Tartary buckwheat varieties were used to investigate the accumulation pattern of the TB02 variety at six different developing stages (10, 13, 16, 20, 25, and 30 DAP). As a result, four different accumulation clusters were obtained for 36 flavonoid components (Figure 5A). Cluster I contained 11 flavonoid components (rutin, astilbin, diosmetin, narirutin, nicotiflorin, baimaside, quercetin, hesperidin, kaempferol, robinin, and homoplantaginin) with levels continuously increasing at 10, 13, 16, 20, and 25 DAP and then decreasing at 30 DAP (Figure 5A). Cluster II consisted of nine flavonoid components (dihydrokaempferol, naringenin chalcone, quercitrin, eriodictyol, catechin gallate, phlorizin, naringenin-7-glucoside, avicularin, and isorhamnetin), which continuously increased from 10 DAP to 16 DAP and then continuously decreased from 20 DAP to 30 DAP (Figure 5A). In cluster III, there were 14 flavonoid components (miquelianin, procyanidin B2, taxifolin, phloretin, epicatechin, catechin, narcissin, isorhamnetin-3-O-neohespeidoside, luteolin, trilobatin, apigenin, astragalin, quercimeritrin, and spiraeoside) with the highest content at 10 DAP and a continuous decrease at the six developing stages (Figure 5A). For cluster IV (orientin and dihydromyricetin), no obvious order was observed (Figure 5A). All of these findings suggest that the accumulation of different flavonoid components has specific and dynamic change patterns during Tartary buckwheat seed development.
To explore the relationship between the content of flavonoid components and the expression of flavonoid biosynthetic structural genes, we investigated the expression patterns of 17 related structural genes in the developing seeds of Tartary buckwheat using seed transcriptome data. As shown in Figure 5B, 17 structural genes were differently expressed during the development of Tartary buckwheat seeds, a result was similar to that previously reported by Li [9]. All 17 structural genes displayed three different expression patterns. The expression pattern of F3′5′H-3, which was a unique member of the first expression pattern, was similar to the accumulation pattern of the flavonoid components in the cluster I (Figure 5A,B). The correlation analysis found that the expression of F3′5′H-3 showed a significantly positive correlation, (r > 0.7, p < 0.05), with the content of the flavonoid components in cluster I (Table S4). Notably, the flavonoid components in the cluster I contained the dominant flavonoid components (rutin, quercetin, nicotiflorin, and kaempferol) in Tartary buckwheat seeds. This suggests that F3′5′H-3 plays an important role in the flavonoid biosynthesis of seeds in Tartary buckwheat and might even be the most crucial gene. In the second expression pattern, ten genes (CHS-1, CHS-2, CHS-4, CHI, F3H-2, FLS, DFR, ANS, LAR, and FtUGT163) presented similar accumulation patterns to the flavonoid components in cluster II (Figure 5A,B), indicating that these ten genes might be major contributors to the accumulation of flavonoid components in cluster II. The third expression pattern contained six genes (CHS-3, F3H-1, F3′H, FtF3′5′H-1, FtF3′5′H-2, and ANR) that displayed similar accumulation patterns to the flavonoid components in the cluster III (Figure 5A,B), suggesting that they might be involved in the accumulation of flavonoid components in cluster III. Notably, the expression of some flavonoid biosynthesis genes showed a significantly positive correlation with the accumulation pattern of flavonoid components, which were directly catalyzed synthesis by these genes’ encoded protein [24,25]. For example, F3H-2 and dihydrokaempferol (r = 0.97, p = 0.0013), F3′H and taxifolin (r = 0.72, p = 0.0121), LAR and catechin (r = 0.87, p = 0.0143), ANR and epicatechin (r = 0.83, p = 0.0040), and so on (Table S4). This indicates that the metabolite biosynthesis genes could be reliably identified though a correlation analysis between the gene expression and metabolite content in different tissues.

3.5. Construction of a Flavonoid Biosynthesis Pathway and Identification of UDP-Glycosyltransferase Genes Involved in the Biosynthesis of Flavonoid Glycosides in Tartary Buckwheat Seeds

To enhance our understanding of flavonoid biosynthesis in Tartary buckwheat seeds, the putative biosynthesis pathways of 41 flavonoid components with the absolute content in seeds of all five Tartary buckwheat were constructed following the flavonoid biosynthesis pathways previously reported in Tartary buckwheat as reference [24,25]. A schematic of the flavonoid biosynthesis pathway in Tartary buckwheat seeds containing 30 major flavonoid components and their absolute quantifications is shown in Figure 6. All of the above not only improved our perception of the flavonoid biosynthesis pathway in Tartary buckwheat seeds but also helped us to identify the genes crucial in flavonoid biosynthesis, which could be applied to increase the flavonoid content in the future.
Among the 30 flavonoid components that were used to construct the flavonoids biosynthesis pathway in Tartary buckwheat seeds, we found that 16 (rutin, narirutin nicotiflorin, baimaside, robinin, orientin, quercitrin, phlorizin, naringenin-7-O-glucoside, avicularin, miquelianin, narcissin, isorhamnetin-3-O-neohesperidoside, astragalin, quercimeritrin, and spiraeoside) were flavonoid glycosides. However, to date, only the glycosyltransferase genes have been identified for rutin and astragalin biosynthesis [20,34]. Considering the 16 flavonoid glycosides expressed differently during Tartary buckwheat seed development, we selected 42 UGTs genes differently expressed in the developing Tartary buckwheat seeds [9] to perform the cluster and correlation analysis with the 16 flavonoid glycosides in the developing seed at six different stages. We found that some UGT genes were clustered with some flavonoid glycosides and that their expression levels displayed a significantly positive correlation (r > 0.7, p < 0.05) with the accumulation pattern of corresponding flavonoid glycosides during Tartary buckwheat seed development (Figure 7 and Table S5). This implied that these UGT genes might be involved in the biosynthesis of their corresponding flavonoid glycosides in Tartary buckwheat seeds but their function needs to be further verified.

4. Conclusions

Sixty-one flavonoid components were absolutely quantified in the seeds of five Tartary buckwheat varieties. The contents of most flavonoid components were significantly different among different varieties or within the same variety. The differences in the content of flavonoid components might be related to the differences in the expression of some flavonoid biosynthesis structural genes in different Tartary buckwheat variety seeds. Among all these determined flavonoid components, rutin, quercetin, nicotiflorin, and kaempferol were the dominant flavonoids in Tartary buckwheat seeds. The accumulation of some flavonoid components displayed a significant positive or negative correlation, implying that their flavonoid biosynthesis in Tartary buckwheat seeds was very complex. Different flavonoid components exhibited specific and dynamic change patterns in the development of Tartary buckwheat seed. Some flavonoid biosynthesis structural genes displayed significantly positive correlations with the accumulation of most flavonoid components in the development of Tartary buckwheat seed, and F3′5′H-3 might be the most crucial contributor to the determination of the flavonoid content in Tartary buckwheat seeds. In addition, some UGT genes might be involved in the biosynthesis of flavonoid glycosides in Tartary buckwheat seeds. All of these provide helpful information for improving our understanding of flavonoid biosynthesis in Tartary buckwheat seeds, and show potential for applications in breeding new cultivars with higher flavonoid contents.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13123006/s1. Table S1: Flavonoid components for which qualitative and absolute quantitative analyses simultaneously performed. Table S2: Primer sequences for qRT-PCR analysis. Table S3: The correlation among 20 flavonoids with the highest contents in Tartary buckwheat seeds. Table S4: The correlation between the accumulation of 36 flavonoids and the expression of structural genes of flavonoid biosynthesis in developing seeds of Tartary buckwheat. Table S5: The correlation between the accumulation of 16 flavonoid glycosides and the expression of 42 UGT genes in developing seeds of Tartary buckwheat.

Author Contributions

J.K.: investigation, methodology, data curation, writing—original draft. B.R.: methodology, investigation, data curation. P.S.: methodology, investigation. Y.C.: methodology, Investigation. Q.C.: funding acquisition, writing—review and editing. H.L.: conceptualization, methodology, funding acquisition, resources, data curation, writing—review and editing, supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (32260461 and 31701494), the Science and Technology Foundation of Guizhou Province (QianKe-HeJiChu-ZK[2021]ZhongDian035), the Key Science and Technology Projects in Yunnan (202202AE090020), the National Natural Science Foundation of China and the Karst Science Research Center of Guizhou province (U1812401) and the Earmarked Fund for China Agriculture Research System (CARS-07-A5).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and the Supplementary Materials.

Conflicts of Interest

The authors declare that they have no known competing financial interest or personal relationship that could have appeared to influence the work reported in this paper.

Abbreviations

AVOVA, one-way analysis of variance; CE, collision energies; CUR, curtain gas; DP, declustering potentials; MRM, multiple reaction monitoring; UHPLC-QqQ-MS, triple quadrupole mass spectrometry; DAP, day after pollination; TB, Tartary buckwheat; IS, internal standards; LIT, linear ion trap; QQQ, triple quadrupole (QQQ); qRT-PCR, quantitative real-time polymerase chain reaction; QTRAP, triple quadrupole-linear ion trap mass spectrometer; UGTs, UDP-glycosyltransferase genes.

References

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Figure 1. The total flavonoid content in the seeds of five Tartary buckwheat varieties. Different letters represent significant difference at p < 0.05.
Figure 1. The total flavonoid content in the seeds of five Tartary buckwheat varieties. Different letters represent significant difference at p < 0.05.
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Figure 2. Venn diagram of the number of absolutely quantified flavonoid components in the seeds of all five Tartary buckwheat varieties. The numbers in brackets for each variety represent the amount of absolutely quantified flavonoid components. The numbers in the diagrams show the common and unique amounts of absolutely quantified flavonoid components among five varieties.
Figure 2. Venn diagram of the number of absolutely quantified flavonoid components in the seeds of all five Tartary buckwheat varieties. The numbers in brackets for each variety represent the amount of absolutely quantified flavonoid components. The numbers in the diagrams show the common and unique amounts of absolutely quantified flavonoid components among five varieties.
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Figure 3. The heat map of the correlation coefficients of the 20 flavonoids with the highest contents in Tartary buckwheat seeds. Red and blue represent the positive and negative correlations, respectively.
Figure 3. The heat map of the correlation coefficients of the 20 flavonoids with the highest contents in Tartary buckwheat seeds. Red and blue represent the positive and negative correlations, respectively.
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Figure 4. Expression of six flavonoid biosynthesis structural genes in the 13 DAP seeds of five Tartary buckwheat varieties. The different letters denote significant differences (p < 0.05).
Figure 4. Expression of six flavonoid biosynthesis structural genes in the 13 DAP seeds of five Tartary buckwheat varieties. The different letters denote significant differences (p < 0.05).
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Figure 5. The accumulation patterns of 36 flavonoid components (A) and the expression patterns of 17 flavonoid biosynthesis structural genes (B) in the developing seeds of Tartary buckwheat.
Figure 5. The accumulation patterns of 36 flavonoid components (A) and the expression patterns of 17 flavonoid biosynthesis structural genes (B) in the developing seeds of Tartary buckwheat.
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Figure 6. The speculative flavonoids biosynthesis pathway in Tartary buckwheat seeds. Red, black, and blue fonts represent the enzymes involved in metabolite synthesis, the flavonoid components not absolutely quantified in this study, and the flavonoid components absolutely quantified in this study, respectively. PAL: phenylalanine ammonia-lyase; C4H: cinnamate-4-hydroxylase; 4CL: 4-coumarate CoA ligase; CHS: chalcone synthase; CHI: chalcone isomerase; F3′H: flavanone-3′-hydroxylase; F3H: flavanone-3-hydroxylase; F3′5′H: flavanone-3′-5′-hydroxylase; FLS: flavonol synthase; UGTs: UDP-glycosyltransferase; CGT: C-glucosyltransferase; OMT: O-methyltransferase; RhaT: rhamnosyltransferase; DFR: dihydroflavonol reductase; ANS: anthocyanin synthase; ANR: anthocyanin reductase; LAR: leucoanthocyanidin reductase.
Figure 6. The speculative flavonoids biosynthesis pathway in Tartary buckwheat seeds. Red, black, and blue fonts represent the enzymes involved in metabolite synthesis, the flavonoid components not absolutely quantified in this study, and the flavonoid components absolutely quantified in this study, respectively. PAL: phenylalanine ammonia-lyase; C4H: cinnamate-4-hydroxylase; 4CL: 4-coumarate CoA ligase; CHS: chalcone synthase; CHI: chalcone isomerase; F3′H: flavanone-3′-hydroxylase; F3H: flavanone-3-hydroxylase; F3′5′H: flavanone-3′-5′-hydroxylase; FLS: flavonol synthase; UGTs: UDP-glycosyltransferase; CGT: C-glucosyltransferase; OMT: O-methyltransferase; RhaT: rhamnosyltransferase; DFR: dihydroflavonol reductase; ANS: anthocyanin synthase; ANR: anthocyanin reductase; LAR: leucoanthocyanidin reductase.
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Figure 7. The clustering heat map of 16 flavonoid glycosides and 42 UGT genes during Tartary buckwheat seed development. The relative content of 16 flavonoid glycosides and the FPKM value of 42 UGT genes were used to construct the clustering heat map.
Figure 7. The clustering heat map of 16 flavonoid glycosides and 42 UGT genes during Tartary buckwheat seed development. The relative content of 16 flavonoid glycosides and the FPKM value of 42 UGT genes were used to construct the clustering heat map.
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Table 1. The absolute content of flavonoid components in the seeds of five Tartary buckwheat varieties (μg g−1 DW; mean ± SD; n = 3). Different letters in the same row show the significant differences (p < 0.05).
Table 1. The absolute content of flavonoid components in the seeds of five Tartary buckwheat varieties (μg g−1 DW; mean ± SD; n = 3). Different letters in the same row show the significant differences (p < 0.05).
No.CompoundsTB01 (μg/g)TB02 (μg/g)TB03 (μg/g)TB04 (μg/g)TB05 (μg/g)
1Rutin12,534.997 ± 270.638 a11,509.258 ± 234.142 b10,988.357 ± 203.185 c11,881.626 ± 265.576 b9021.432 ± 227.818 d
2Quercetin1083.64 ± 24.935 d1494.262 ± 75.715 b1392.312 ± 20.082 bc1346.971 ± 20.714 c2290.068 ± 101.593 a
3Nicotiflorin543.077 ± 17.839 d1016.949 ± 27.376 a711.654 ± 18.748 c750.712 ± 32.807 bc766.635 ± 17.438 b
4Kaempferol75.353 ± 7.239 c136.044 ± 6.514 ab114.235 ± 5.483 b113.818 ± 10.216 b142.295 ± 24.371 a
5Quercimeritrin102.863 ± 2.567 a45.01 ± 7.45 b23.859 ± 2.450 c16.605 ± 1.075 c45.383 ± 4.918 b
6Hesperidin16.393 ± 1.303 a15.980 ± 0.87 a16.253 ± 1.465 a16.427 ± 1.23 a17.543 ± 3.263 a
7Procyanidin B29.835 ± 0.841 b10.338 ± 1.215 b16.530 ± 1.177 a15.674 ± 0.918 a6.226 ± 1.049 c
8Epicatechin8.428 ± 0.523 c6.820 ± 0.891 d20.496 ± 1.033 a12.309 ± 0.685 b5.393 ± 0.231 d
9Narcissin5.486 ± 0.08 b5.828 ± 0.138 ab6.189 ± 0.276 a4.578 ± 0.263 c2.909 ± 0.169 d
10Catechin2.586 ± 0.199 a2.049 ± 0.211 b2.053 ± 0.207 b2.069 ± 0.135 b1.429 ± 0.154 c
11Astragalin2.274 ± 0.325 b3.183 ± 0.057 a3.125 ± 0.312 a1.746 ± 0.051 c1.42 ± 0.039 c
12Baimaside2.257 ± 0.048 a0.866 ± 0.102 b0.605 ± 0.059 c0.876 ± 0.063 b0.653 ± 0.028 c
13Spiraeoside1.247 ± 0.188 a0.074 ± 0.008 bc0.032 ± 0.0006 c0.154 ± 0.014 bc0.208 ± 0.032 b
14Catechin gallate0.628 ± 0.022 b0.250 ± 0.016 c0.759 ± 0.122 b1.137 ± 0.089 a0.304 ± 0.063 c
15Taxifolin0.558 ± 0.037 a0.457 ± 0.013 b0.173 ± 0.011 d0.091 ± 0.003 e0.253 ± 0.008 c
16Phlorizin0.554 ± 0.053 a0.581 ± 0.056 a0.516 ± 0.037 a0.314 ± 0.016 c0.432 ± 0.013 b
17Avicularin0.477 ± 0.038 a0.397 ± 0.009 b0.302 ± 0.024 c0.263 ± 0.021 cd0.230 ± 0.019 d
18Naringenin-7-glucoside0.371 ± 0.006 a0.322 ± 0.015 b0.159 ± 0.013 c0.093 ± 0.012 d0.144 ± 0.025 c
19Isorhamnetin0.271 ± 0.009 c0.430 ± 0.011 a0.393 ± 0.009 b0.258 ± 0.034 c0.175 ± 0.011 d
20Quercitrin0.297 ± 0.027 b0.339 ± 0.019 a0.185 ± 0.012 c0.170 ± 0.006 c0.262 ± 0.029 b
21Miquelianin0.195 ± 0.0026 a0.065 ± 0.001 b0.018 ± 0.001 e0.029 ± 0.002 d0.034 ± 0.003 c
22Astilbin0.124 ± 0.0031 b0.154 ± 0.013 a0.047 ± 0.004 d0.047 ± 0.009 d0.063 ± 0.004 c
23Dihydrokaempferol0.058 ± 0.042 ab0.080 ± 0.008 a0.063 ± 0.003 ab0.036 ± 0.004 b0.063 ± 0.001 ab
24Robinin0.095 ± 0.0021 a0.035 ± 0.004 c0.0064 ± 0.001 e0.016 ± 0.001 d0.058 ± 0.001 b
25Isorhamnetin-3-O-neohespeidoside0.069 ± 0.012 b0.116 ± 0.016 a0.059 ± 0.008 b0.072 ± 0.009 b0.051 ± 0.003 b
26Orientin0.075 ± 0.0044 a0.076 ± 0.001 a0.072 ± 0.002 a0.072 ± 0.001 a0.075 ± 0.002 a
27Scutellarin0.055 ± 0.0068 a0.035 ± 0.002 b0.019 ± 0.002 c0.022 ± 0.002 c0.033 ± 0.004 b
28Hydroxysafflor yellow A0.063 ± 0.0066 a0.042 ± 0.006 b0.059 ± 0.007 a0.043 ± 0.008 b0.025 ± 0.002 c
29Naringenin chalcone0.055 ± 0.0032 b0.217 ± 0.014 a0.041 ± 0.003 c0.026 ± 0.001 d0.055 ± 0.005 b
30Phloretin0.046 ± 0.0044 a0.026 ± 0.003 c0.018 ± 0.001 d0.033 ± 0.002 b0.026 ± 0.003 c
31Narirutin0.047 ± 0.0012 b0.063 ± 0.011 a0.014 ± 0.003 c0.012 ± 0.002 c0.019 ± 0.0006 c
32Eriodictyol0.042 ± 0.0021 c0.773 ± 0.078 a0.052 ± 0.008 c0.025 ± 0.003 c0.548 ± 0.068 b
33Luteolin0.015 ± 0.0029 a0.016 ± 0.001 a0.011 ± 0.0006 b0.0084 ± 0.0011 bc0.0059 ± 0.001 c
34Dihydromyricetin0.056 ± 0.0131 ab0.098 ± 0.007 a0.028 ± 0.004 b0.058 ± 0.002 ab0.025 ± 0.001 b
35Trilobatin0.014 ± 0.0021 c0.02 ± 0.002 b0.029 ± 0.004 a0.016 ± 0.0023 bc0.012 ± 0.001 c
36Baicalein0.012 ± 0.0012 a0000
37Liquiritigenin0.0089 ± 0.0007 cd0.0076 ± 0.0006 d0.022 ± 0.002 a0.013 ± 0.002 b0.012 ± 0.02 bc
38Galangin0.0093 ± 0.0011 a0.0076 ± 0.0014 b000
39Homoplantaginin0.0075 ± 0.0013 a0.0059 ± 0.001 b0.0048 ± 0.0002 b0.0025 ± 0.0003 c0.0007 ± 0.0001 d
40Apigenin0.0037 ± 0.0012 a0.005 ± 0.0009 a0.0008 ± 0.0009 b0.0008 ± 0.0001 b0.0046 ± 0.0008 a
41Diosmetin0.0025 ± 0.0004 a0.0003 ± 0.0001 c0.0003 ± 0.0001 c0.0005 ± 0.0001 c0.0021 ± 0.00001 b
42Pinocembrin0.0032 ± 0.0006 a0000
43Isoliquiritigenin0.0013 ± 0.0006 ab0.0011 ± 0.0002 bc0.0018 ± 0.0002 a0.0007 ± 0.0002 c0.001 ± 0.0002 bc
44Isosilybin0.0014 ± 0.0003 b00.0013 ± 0.0002 b0.0009 ± 0.0002 c0.0028 ± 0.0003 a
45Scutellarein tetramethyl ether0.0002 b0.0003 ± 0.0001 b0.0014 ± 0.0001 a0.0003 ± 0.0001 b0.0014 ± 0.0001 a
46Neohesperidin dihydrochalcone00.045 ± 0.006 a000
47Gallocatechin gallate00.051 ± 0.005 b0.057 ± 0.003 b0.043 ± 0.009 b0.106 ± 0.013 a
48Isosakuranetin00000.0012 ± 0.002 a
49Sakuranetin00.002 ± 0.0003 a000.0008 ± 0.0004 b
505-O-Demethylnobiletin000.0012 ± 0.0003 b0.0008 ± 0.0001 b0.0043 ± 0.0004 a
51Tangeretin00.0012 ± 0.0002 b0.0021 ± 0.0003 b00.036 ± 0.0035 a
52Nobiletin00.0009 ± 0.0002 b0.0021 ± 0.0003 b00.0227 ± 0.0021 a
53Baicalin00.0294 ± 0.009 a000
547,4′-Di-O-methylapigenin00000.0011 ± 0.0002 a
55Sinensetin00000.0009 ± 0.0001 a
56Chrysin00.0002 ± 0.0001 a000
57Acacetin00000.0016 ± 0.001 a
58Genkwanin00000.0034 ± 0.0005 a
59Afzelin00.0022 ± 0.0002 a0.0016 ± 0.0003 b00
603,7,4′-Trihydroxyflavone00.2520 ± 0.048 a000
61Glycitin000.144 ± 0.012 a00
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Ke, J.; Ran, B.; Sun, P.; Cheng, Y.; Chen, Q.; Li, H. An Evaluation of the Absolute Content of Flavonoids and the Identification of Their Relationship with the Flavonoid Biosynthesis Genes in Tartary Buckwheat Seeds. Agronomy 2023, 13, 3006. https://doi.org/10.3390/agronomy13123006

AMA Style

Ke J, Ran B, Sun P, Cheng Y, Chen Q, Li H. An Evaluation of the Absolute Content of Flavonoids and the Identification of Their Relationship with the Flavonoid Biosynthesis Genes in Tartary Buckwheat Seeds. Agronomy. 2023; 13(12):3006. https://doi.org/10.3390/agronomy13123006

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Ke, Jin, Bin Ran, Peiyuan Sun, Yuanzhi Cheng, Qingfu Chen, and Hongyou Li. 2023. "An Evaluation of the Absolute Content of Flavonoids and the Identification of Their Relationship with the Flavonoid Biosynthesis Genes in Tartary Buckwheat Seeds" Agronomy 13, no. 12: 3006. https://doi.org/10.3390/agronomy13123006

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