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Differences in Cotyledon Color and Harvest Period Affect the Contents of Major Isoflavones and Anthocyanins in Black Soybeans
Plant Breed. Biotech. 2021;9:65-76
Published online March 1, 2021
© 2021 Korean Society of Breeding Science.

Yu-Mi Choi1, Hyemyeong Yoon1, Myoung-Jae Shin1, Yoonjung Lee1, Sukyeung Lee1, On Sook Hur1, Na Young Ro1, Ho-Cheol Ko2, Jeongyoon Yi1, Sang Hoon Lee3, Heon-Woong Kim3, Yu Jin Hwang3, Myung-Chul Lee1, Kebede Taye Desta1,4,*

1National Agrobiodiversity Center, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju 54874, Korea
2Rural Development Administration, Jeonju 54875, Korea
3Department of Agro-Food Resources, National Institute of Agricultural Sciences, Wanju 54874, Korea
4Department of Applied Chemistry, Adama Science and Technology University, Adama 1888, Ethiopia
Corresponding author: Kebede Taye Desta, kebetila@gmail.com, Tel: +82-63-238-4864, Fax: +82-63-238-4909
Received November 24, 2020; Revised January 14, 2021; Accepted February 16, 2021.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Several environmental and genetic factors affect the isoflavone and anthocyanin contents in soybeans. This study aimed to assess the influences of cotyledon color and harvest period on the contents of five major isoflavones and three major anthocyanins in 323 black soybean landraces grown in Korea. In all the soybeans, malonylgenistin, malonyldaidzin and cyanidin-3-O-glucoside were the foremost components. The total isoflavone (TIC) and total anthocyanin (TAC) contents were in the ranges of 491.29-1998.39 μg/g and 452.60-2789.37 mg/100 g on dry weight basis, respectively. Both the average TIC and TAC were higher in green cotyledon soybeans (1493.93 μg/g and 1656.05 mg/100 g, respectively) than in yellow cotyledon soybeans (1423.09 μg/g and 1489.30 mg/100 g, respectively). With respect to the harvest period, the average TIC was in the order of Late-October (1517.31 μg/g) > Mid-October (1454.54 μg/g) > Early-October (1340.95 μg/g). Moreover, the average TAC decreased in the order of Late-October (1765.76 mg/100 g) > Mid-October (1503.93 mg/100 g) > Early-October (1350.91 mg/100 g). In general, cotyledon color appeared to cause a significant variation on TAC (P < 0.05) but not on TIC, whereas the harvest period appeared to cause significant variations on both the TAC and TIC. Among the 323 landraces, 20 were identified to contain high TIC (> 1800 μg/g) and TAC (> 2000 mg/100 g) in their seeds and hence, could be considered as important sources of dietary isoflavones and anthocyanins. Besides, they possibly provide a wide spectrum of options if considered during the development of improved soybean genotypes.
Keywords : Anthocyanin, Cotyledon color, Glycine max, Harvest period, Isoflavone, Soybean
INTRODUCTION

Soybeans (Glycine max L. (Merrill)) are rich sources of proteins, fatty acids, vitamins, and minerals (Isanga and Zhang 2008; Shi et al. 2010). Besides, soybeans are known for their non-nutritive components including phenolic acids, isoflavones, and anthocyanins. These metabolites are recognized to have health-promoting properties and play important roles in preventing many ailments such as cancer, inflammation, obesity, and diabetes (Jian 2009; Gansen and Xu 2017).

Soybean isoflavones are structurally diverse and found in aglycone (daidzein, genistein, glycitein), b-glycoside (daidzin, genistin, glycitin), Omalonylglycoside (malonyldaidzin, malonylgenistin, malonylglycitin), and Oacetylglycoside (acetyldaidzin, acetylgenistin, acetylglycitin) forms (Lin and Guisti 2005; Cho et al. 2013). On the other hand, several anthocyanins including cyanidin, delphinidin, peonidin, petunidin, malvidin, and pelargonidin have been identified both in their aglycone and glycosylated forms, mainly in black soybeans (Choung et al. 2001; Zhang et al. 2011; Cho et al. 2013). Comparable studies revealed that the malonylated and b-glycoside forms are the most abundant isoflavones. Besides, cyanidin-3-O-glucoside (C-3-O-G), delphinidin-3-O-glucoside (D-3-O-G), and petunidin-3-O-glucoside (Pt-3-O-G) are the most abundant anthocyanins in black soybeans (Choung et al. 2001; Lin and Guisti 2005). Such differences in the bioavailability of isoflavones and anthocyanins could be attributable to the variations in several major and minor genes and quantitative trait loci that regulate their biosynthetic pathways (Watanabe et al. 2002; Ahmad et al. 2017; Wu et al. 2020).

It is widely documented that the contents of antho-cyanins and isoflavones in soybeans are affected by seed-related agronomical characters and environmental factors (Hoeck et al. 2000; Murphy et al. 2002; Riedl et al. 2007; Carrera et al. 2011). On the other hand, there is a growing interest in the development of improved soybean cultivars through breeding and genetic engineering (Stewart-Brown et al. 2019; Jang and Lee 2020). Therefore, studies that investigate the influences of genetic and environmental factors on the contents of soybean metabolites are receiving much attention and are becoming persistently necessary. Besides, such studies provide vital information to distinguish the best genotypes among a large population of genetic resources (Cober and Voldeng 2000; Dubey et al. 2019; Li et al. 2020).

In Korea, black soybeans are largely consumed and many researchers determined the levels of dietary isoflavones and anthocyanins in their seeds. Moreover, the influences of environmental factors such as place of cultivation, planting dates and growing season, and seed-related characters such as seed coat color, maturity and seed weight have been investigated (Lee et al. 2003; Ha et al. 2009; Kim et al. 2012a, 2012b; Kim et al. 2014; Le et al. 2020). Many of these studies, however, conducted a separate analysis and/or covered only a few soybean genetic materials. Besides, studies that assess the influences of cotyledon color and harvest period are still limited (Kim et al. 2005; Lee et al. 2010). To view the effects of genetic and environmental factors in wider aspects, studies that encompass a large population of soybean genetic materials are still desired. This study aimed to determine the contents of five major isoflavones (daidzin, genistin, glycitin, malonyldaidzin, and malonylgenistin) and three major anthocyanins (C-3-O-G, D-3-O-G, and Pt-3-O-G) in 323 black soybean landraces recently cultivated in Korea and assess if each content is related to the difference in cotyledon color and harvest period.

MATERIALS AND METHODS

Chemicals and reagents

Anthocyanin standards including D-3-O-G and C-3-O-G were obtained from Caymanchem (Ann Arbor, MI, USA) while Pt-3-O-G was purchased from Carbosynth (Berkshire, UK). The isoflavone standards including daidzin, glycitin, genistin, malonyldaidzin, and malonylgenistin, and HPLC-grade solvents including water, acetonitrile, methanol, hydrochloric acid, and formic acid were purchased from Sigma Aldrich (St. Louis, MO, USA). All the chemicals and reagents were of analytical grade and were directly used without further purification.

Plant materials

The 323 black soybean landraces of Korean origin were obtained from the Gene bank of the National Agrobiodiversity Center, Rural Development Administration (RDA, Jeonju, Korea). The soybeans were directly sown at a spacing of 90 cm in an experimental field located at the center on June 5, 2018. All landraces were cultivated under similar conditions and the average temperature and precipitation during the cropping period are shown in Table 1. The change in pod color was used as an index of maturity, and seeds were harvested when 95% of their pods attained matured color (Zhou et al. 2019). The harvest period spanned from the beginning to the end of October 2018, and soybeans were grouped based on their harvest period as Early-October (Oct. 1-10, 72 landraces), Mid-October (Oct. 11-20, 155 landraces), and Late-October (Oct. 21-31, 96 landraces). Besides, the soybeans were grouped as green (113 landraces) and yellow (210 landraces) based on their cotyledon color (Fig. 1). Whole seed samples from each category were dried in a Bionex oven (Vision Scientific, Daejeon, Korea) for three days at 50℃, powdered, and used for isoflavone analysis, whereas seed coat samples were lyophilized in an LP500 freeze drier (ilShinBioBase, Dongducheon, Korea), powdered, and used for anthocyanin analysis. For ease of presentations, the samples were coded based on the appearance of their introduction (IT) number. The sample code, IT number, cotyledon color, days to maturity, and harvest period for each of the soybean landraces are summarized in Supplementary Table S1.

Table 1 . Average temperature and precipitation of the cultivation area during the cropping period (2018).

ParameterCultivation periodHarvest period


JuneJulyAugustSeptemberEarly-October (1-10)Mid-October (11-20)Late-October (21-31)
Average temperature (℃)23.127.828.621.716.11312.1
Average precipitation (mm)137.2169.1368.9101.898.70.923.4

Figure 1. The frequency of black soybeans with respect to cotyledon color (A) and harvest period (B).

Extraction of isoflavone and anthocyanin

Isoflavones were extracted using the method developed by Nawaz et al. (2018) with some modification. Initially, 20 mg of powdered seed sample was added into an extraction tube followed by the addition of 3 mL of methanol. The mixture was then sonicated for 20 minutes in a water bath, shaken, and centrifuged at 4000 rpm for 10 minutes. The upper supernatant was retained and filtered through a 0.2 µm syringe filter into a glass vial for HPLC analysis. Anthocyanin extraction was performed according to the method reported by Choung et al. (2001). Briefly, 200 mg of powdered soybean seed coat sample was placed in a 50 mL capacity extraction tube. Then, 30 mL of 80% methanol containing 1% HCl was added and the mixture was sonicated for 10 minutes followed by hydrolyzation in a water bath for 20 minutes. The mixture was then taken off and incubated at 65℃ with instant shaking (280 rpm). Then, the mixture was cooled in an ice bath for 15 minutes and centrifuged at 1000 rpm for 4 minutes. The upper supernatant was taken, filtered through a 0.45 µm micro-membrane into an injection vial, and readied for HPLC analysis.

HPLC analysis of isoflavones and anthocyanins

Identification and quantification of the target isoflavones and anthocyanins were achieved using the corresponding external standards. Analysis was conducted on a Nanospace SI-2 Semi-microcolumn HPLC system (Shiseido, Tokyo, Japan) coupled to a UV-Vis detector. Isoflavones were eluted on a Shiseido UG-120 column (4.6 × 250 mm; 5 µm) which was maintained at 30℃ while anthocyanins were separated on a Shiseido C18 column type MGII (4.6 × 250 mm; 5 µm) which was maintained at 40℃. During isoflavone analysis, a binary solvent system composed of water (A) and acetonitrile (B) was used as a mobile phase. The gradient elution was started with 15%B for the first 10 minutes followed by an increase to 20%B for another 10 minutes, and to 50% for 20 minutes. Then, the mobile phase was equilibrated to 15%B for the last 10 minutes. The solvent flow rate was 0.8 mL/minute throughout the analysis and the sample injection volume was 10 µL. The isoflavones were detected at 260 nm wavelength in the acquired chromatograms. During anthocyanin analysis, the mobile phase was composed of water (A) and acetonitrile (B) each containing 5% formic acid. The elusion was started with 10%B followed by an increase to 15%B at 0.8 mL/minute flow rate until 10 minutes, then to 46%B at 0.8 mL/minute until 15.3 minutes, and to 90%B at 1 mL/minute until 17 minutes. Finally, the solvent was equilibrated to 10%B at 0.8 mL/minute until 24 minutes. The sample injection volume was 20 µL and the anthocyanins were read at 520 nm wavelength.

Statistical analysis

All measurements in the present study were carried out in duplicates, and results were expressed as mean ± standard deviation (SD) on a dry weight basis. One-way analysis of variance was computed using xlstat-software (Addinsoft, NY, USA) and applied to statistically determine the difference between treatments. Box plots, principal component and Pearson correlation analysis were performed on R-software version 4.0.2 (http://www.r-project.org/).

RESULTS

Distribution and content of isoflavones and anthocyanins

The individual isoflavones and anthocyanins were identified and quantified using the corresponding external standards as described before. All five isoflavones (daidzin, glycitin, genistin, malonyldaidzin, and malonylgensitin) were detected in every landrace. With respect to individual anthocyanins, however, variations were observed for few landraces. C-3-O-G was detected in all landraces, whereas D-3-O-G and Pt-3-O-G were not detected in 22 and 27 landraces, respectively. All the landraces that did not contain D-3-O-G also did not contain Pt-3-O-G.

The soybean landraces showed variations in both isoflavone and anthocyanin contents (Supplementary Table S1). The total isoflavone (TIC) and total anthocyanin (TAC) contents were in the ranges of 491.29-1998.39 µg/g and 452.60-2789.37 mg/100 g with means of 1447.87 µg/g and 1547.64 mg/100 g, respectively. Compared to the TIC, the TAC had a higher coefficient of variation (%CV) (Supplementary Table S2). The highest TIC was found in landrace S25 (IT: 121504) and was approximately 4-times higher than the lowest TIC found in landrace S84 (IT: 177871) (P < 0.05). Besides, the highest TAC found in landrace S191 (IT: 242611) was nearly 8-fold higher than the lowest TAC found in landrace S45 (IT: 177226) (P < 0.05). In general, 17.03% of the soybean landraces had a TIC > 1800 µg/g, and out of these 36.36% had a TAC > 2000 mg/100 g each being much higher than the observed average values. Among individual isoflavones, malonly-genistin was the foremost component with a mean of 778.76 µg/g followed by malonyldaidzin that had a mean content of 396.05 µg/g. The former contributed between 34.21 and 74.00% to the TIC while the latter accounted for between 8.94 and 41.61% of the TIC. On the other hand, glycitin was the least concentrated isoflavone with a mean of 30.81 µg/g. The average daidzin and genistin contents were 93.07 and 149.18 µg/g, respectively. With respect to anthocyanins, C-3-O-G content was the highest in all the landraces with a mean of 1197.46 mg/100 g and contributed between 31.26 and 100% of the TAC. Moreover, the average D-3-O-G and Pt-3-O-G contents were 265.86 and 84.31 mg/100 g, respectively.

Influence of cotyledon color on isoflavone and anthocyanin contents

The box plots in Fig. 2 show the variations in isoflavone and anthocyanin contents among the green and yellow cotyledon black soybeans. The corresponding numerical values can be viewed in Supplementary Table S2. The average TIC was higher in green cotyledon soybeans (1499.93 µg/g) than in yellow cotyledon soybeans (1423.09 µg/g). Besides, all individual isoflavones, except glycitin, followed a similar pattern of variation as the TIC each being higher in green cotyledon soybeans than in yellow cotyledon soybeans. Among the five isoflavones, the level of malonylgenistin was the highest in each class. The average TAC followed a comparable pattern as the TIC and was higher in green cotyledon soybeans (1656.05 mg/100 g) than in yellow cotyledon soybeans (1489.30 mg/100 g). Among individual anthocyanins, C-3-O-G and Pt-3-O-G contents showed a comparable pattern as the TAC and each was higher in green cotyledon soybeans (1311.52 and 90.79 mg/100 g, respectively) than in yellow cotyledon soybeans (1136.08 and 80.83 mg/100 g, respectively). Unlike these, yellow cotyledon soybeans had a higher average D-3-O-G content (272.38 mg/100 g) than green cotyledon soybeans (253.75 mg/100 g).

Figure 2. Influence of cotyledon color on the contents of isoflavones and anthocyanins in 323 black soybean landraces cultivated in Korea. C-3-O-G: Cyanidin-3-O-glucoside; D-3-O-G: Delphinidin-3-O-glucoside; Pt-3-O-G: Petunidin-3-O-glucoside; TAC: Total anthocyanin content. TIC: Total isoflavone content. Different letters on box plots in a category represent means that are significantly different (P < 0.05).

Relationship between harvest period and isoflavone and anthocyanin contents

The relationship between harvest periods (Early-October, Mid-October and Late-October) and isoflavone and anthocyanin contents are displayed in Fig. 3. The corresponding numerical values can be viewed in Supplementary Table S2. The average TIC was the highest in Late-October (1517.31 µg/g) followed by Mid-October (1454.54 µg/g) and Early-October (1340.95 mg/g). Among individual isoflavones, the average genistin and malonylgenistin contents were the highest in Late-October (166.84 and 828.72 mg/g, respectively) followed by Mid-October (147.31 and 764.92 mg/g, respectively) and Early-October (129.66 and 741.95 mg/g, respectively). Besides, the average daidzin and malonyldaidzin contents were in the order of Mid-October (98.26 and 412.02 mg/g, respectively) > Late-October (92.81 and 400.29 mg/g, respectively) > Early-October (82.26 and 356.01 mg/g, respectively) while the average glycitin content was in the order of Mid-October (32.03 mg/g) > Early-October (31.08 mg/g) > Late-October (28.63 mg/g). With respect to anthocyanins, the average TAC followed a similar pattern of variation as the TIC and was the highest in Late-October (1765.76 mg/100 g) than in Mid-October (1503.93 mg/100 g) and Early-October (1350.91 mg/100 g). Moreover, the average content of the principal anthocyanin, C-3-O-G, also decreased in the order of Late-October (1444.26 mg/100 g) > Mid-October (1132.40 mg/100 g) > Early-October (1008.46 mg/100 g). The average D-3-O-G and Pt-3-O-G contents were the highest in Mid-October (281.30 and 90.23 mg/100 g, respectively) each followed by Early-October (274.32 mg/100 g) and Late-October (86.89 mg/100 g), respectively.

Figure 3. Relationship between harvest period and the contents of isoflavones and anthocyanins in 323 soybean landraces cultivated in Korea. C-3-O-G: Cyanidin-3-O-glucoside; D-3-O-G: Delphinidin-3-O-glucoside; Pt-3-O-G: Petunidin-3-O-glucoside; TAC: Total anthocyanin content. TIC: Total isoflavone content. Different letters on box plots in a category represent means that are significantly different (P < 0.05).

Correlation and principal component analysis

The pairwise association between isoflavones and anthocyanins in the entire soybean landraces was computed using Pearson correlation analysis (Table 2). The TIC was positively correlated with all the individual isoflavones, and all associations were significant. The TIC showed a strong association with malonylglycitin (r = 0.90) and malonyldaidzin (r = 0.81), the two abundant isoflavones. Besides, the individual isoflavones were positively associated with each other. Likewise, the TAC was positively correlated to all individual anthocyanins, and showed a strong association with the major anthocyanin, C-3-O-G (r = 0.95). Some of the individual isoflavones including malonylgenistin and genistin showed positive associations with other individual anthocyanins including C-3-O-G and D-3-O-G. However, the correlation coefficient values were far from +1. The distribution of the soybean landraces based on the isoflavone and anthocyanin contents was further viewed by principal component analysis (PCA). Four components had eigenvalues of >1 and explained about 79.92% of the total variance (Table 3). The first two components alone explained 54.41% of the total variance and hence, were considered for analysis. The first principal component (PC1) accounted for 34.31% of the variance while the remaining 20.10% was contributed by the second principal component (PC2). The soybean landraces were widely dispersed in the score plot by cotyledon color (Fig. 4A) and harvest period (Fig. 4B). All isoflavones except glycitin were the main factors for the variation observed along PC1, whereas C-3-O-G was the major contributor for the variation observed along PC2 (Fig. 4C). The contributions from malonyldaidzin (17.93%) and malonylgenistin (17.89%) were comparable with each other. Besides, the b-glycoside derivatives of these isoflavones, genistin (14.99%) and daidzin (14.88%), were also comparable with each other. The least concentrated isoflavone (glycitin) contributed only 1.17% along PC1. On the other hand, C-3-O-G contributed 42.39% along PC2. The contributions of D-3-O-G and Pt-3-O-G were minimal at 0.08 and 1.17%, respectively. Notably, the strong associations of TIC and TAC with their respective major components were noted in the loading plot (Fig. 4C).

Table 2 . Pearson correlation coefficient (r) for the pairwise association between isoflavones and anthocyanins.

VariablesDaidzinGlycitinGenistinMalonyl-daidzinMalonyl-genistinTICD-3-O-GC-3-O-GPt-3-O-G
Glycitin0.27****
Genistin0.69****0.22****
Malonyldaidzin0.61****0.08ns0.25****
Malonylgenistin0.22****0.01ns0.43****0.57****
TICz)0.58****0.14*0.59****0.81****0.90****
D-3-O-Gy)0.04ns0.13*0.02ns0.02ns‒0.04ns‒0.01ns
C-3-O-Gx)‒0.02ns‒0.16**0.12*0.01ns0.20***0.17**‒0.05ns
Pt-3-O-Gw)0.03ns0.11*0.19**0.01ns0.14*0.13*0.40****‒0.14*
TACv)‒0.01ns‒0.12*0.14*0.11ns0.20**0.18**0.24****0.95****0.05ns

z)Total isoflavone content; y)Delphinidin-3-O-glucoside; x)Cyanidin-3-O-glucoside; w)Petunidin-3-O-glucoside; v)Total anthocyanin content.

nsNot significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.


Table 3 . Contributions and eigenvalues of variables in the first four principal components.

VariablesPrincipal components

PC1PC2PC3PC4
Daidzin14.885.610.0616.36
Glycitin1.178.236.2926.27
Genistin14.991.170.7711.48
Malonyldaidzin17.930.562.643.31
Malonylgenistin17.890.211.1318.82
Total isoflavone content26.650.271.034.45
Delphinidin-3-O-glucoside0.150.0845.980.93
Cyanidin-3-O-glucoside2.4942.390.084.73
Petunidin-3-O-glucoside0.871.1737.9511.17
Total anthocyanin content2.9940.304.082.48
Eigenvalue3.442.001.491.06
Variability (%)34.3120.1014.9010.61
Cumulative variance (%)34.3154.4169.3179.92

Figure 4. Score plots of 323 black soybean landraces over cotyledon color (A) and harvest period (B) and loading plots of variables (C) obtained from principal component analysis based on isoflavone and anthocyanin contents. C-3-O-G: Cyanidin-3-O-glucoside; D-3-O-G: Delphinidin-3-O-glucoside; Pt-3-O-G: Petunidin-3-O-glucoside; TAC: Total anthocyanin content; TIC: Total isoflavone content.
DISCUSSION

In this study, 323 black soybean landraces were grown in Korea, the contents of three common anthocyanins (D-3-O-G, C-3-O-G, and Pt-3-O-g) and five common isoflavones (daidzin, glycitin, genistin, malonyldaidzin, and malonylgenistin) were analyzed, and the association of each with cotyledon color and harvest period was assessed.

The TIC and TAC were in the ranges of 491.29-1998.39 mg/g and 452.60-2789.37 mg/100 g, respectively. Of the entire soybeans analyzed in the present study, 17.03% of the landraces (i.e. 55 landraces) had a TIC of > 1800 mg/g, and out of these 36.36% (i.e. 20 landraces) had a TAC of > 2000 mg/100 g (Supplementary Table S1). Hence, these landraces could be important sources of high isoflavone and anthocyanin concentrations. Previously, many studies determined the TIC and TAC in various Korean black soybeans. Nevertheless, the reported total contents are inconsistent and wide-ranging due to the disparity in the number and identity of target components (Kim et al. 2012a, 2012b; Lee et al. 2020). Among individual isoflavones, malonylgenistin and malonyldaidzin were the major components in all the landraces. Earlier studies also noted the dominance of these isoflavones, mainly in unprocessed seeds (Ha et al. 2009; Kim et al. 2012b; Bursać et al. 2017; Wu et al. 2017). A genomic study conducted by Ahmad et al. (2017) directed that the abundance of specific enzymes including uridine diphosphate-dependent glycosyltransferase and malonyl-Co-A dependent acyltransferase could favor the high accumulation of malonylated isoflavones in soybeans. Besides, C-3-O-G was the foremost anthocyanin in all the landraces, and the finding was consistent with previous reports (Zhang et al. 2011; Cho et al. 2013; Koh et al. 2014; Lee et al. 2020).

In their early growth seedling, black soybeans develop either green or yellow cotyledon which could later affect the metabolite contents in matured seeds (Eum et al. 2020). In the present study, 113 landraces developed green cotyledon while the remaining 210 landraces developed yellow cotyledon. Green cotyledon soybeans had a higher average TIC than yellow cotyledon soybeans although the variation was not significant (P > 0.05) (Fig. 2). With respect to individual isoflavones, the contents of all except glycitin were higher in green than in yellow cotyledon soybeans. Nevertheless, only the variations in genistin and glycitin contents were significantly different (P < 0.05). To the best of our knowledge, only two studies by Kim et al. (2005) and Lee et al. (2010) previously attempted to analyze the isoflavone content in soybeans with respect to cotyledon color. Kim et al. (2005) analyzed three isoflavones (aglycones) in 59 black soybeans and found no significant variation in the TIC between green and yellow cotyledon soybeans. Moreover, Lee et al. (2010) analyzed 12 isoflavones in 268 soybeans of different seed coat colors and failed to observe significant variation in the TIC between green and yellow cotyledon soybeans. These findings were in support of our observations. In general, cotyledon color appeared to have no significant effect on the TIC in black soybeans. Thence, it can be suggested that soybean genotypes should not be selected based on the color of their cotyledon for their isoflavone concentration. With respect to anthocyanins, the average TAC was high in green cotyledon soybeans compared to yellow cotyledon soybeans. Unlike the average TIC, the variation of the average TAC between the green and yellow cotyledon soybeans was significantly different (P < 0.05) (Fig. 2). Besides, C-3-O-G and Pt-3-O-G contents were higher in green cotyledon soybeans while D-3-O-G content was higher in yellow cotyledon soybeans. The variation in C-3-O-G content between green and yellow cotyledon soybeans was significantly different (P < 0.05). Our results are in agreement with the findings by Kim et al. (2005) who similarly analyzed these three anthocyanins with respect to cotyledon color in black soybeans. In general, black soybeans that develop green cotyledon in the course of their early growth could be important sources of a high level of dietary anthocyanin.

In Korea, the fall season (September to November) is the harvesting time for most summer cropping cereals and legumes (Wang and Mauzerall 2004). In this study, the harvest period ranged from the beginning to the end of October. Of the entire 323 soybeans, 72 landraces were harvested in Early-October, 155 landraces in Mid-October, and 96 landraces in Late-October (Fig. 1). The days to maturity was in the ranges of 116-146 days with an average of 122, 130, and 141 days for landraces harvested in Early-October, Mid-October, and Late-October, respectively. Harvest period appeared to cause significant variation in the contents of isoflavones and anthocyanins. Both the average TIC and TAC were the highest in Late-October followed by Mid-October and Early-October. The average TIC in Late-October and Mid-October was significantly different from the TIC in Early-October (P < 0.05), but not with each other (Fig. 3). Moreover, the variation in the TAC was significantly different among the three harvest periods (P < 0.05). Variations were also observed with respect to individual components (Fig. 3). The contents of malonylgenistin, malonyldaidzin and C-3-O-G were each the highest in Late-October (P < 0.05). Previously, many researchers also noted variations in the level of isoflavones in other legumes and fruits at different harvest periods (Ribeiro et al. 2007; Winardiantika et al. 2015; Mustonen et al. 2018). These studies attested to the impacts of temperature, duration and intensity of sunshine, pre-cipitation, and rainfall on metabolite contents. Moreover, it was verified that a relatively longer cultivation period promotes the accumulation of more isoflavone and anthocyanin in soybeans which was consistent with our observations (Wang et al. 2000; Joo et al. 2004; Azam et al. 2020). In general, our findings suggest that Late-October could be an ideal period in Korea to harvest isoflavone and anthocyanin-rich soybeans.

The pair-wise correlation between the different components and the distributions of the soybean landraces were viewed by Pearson correlation and principal component analysis. The TAC and TIC showed strong associations with their respective major components, and the observations were comparable with many previous studies (Bursac et al. 2017; Wu et al. 2017; Azam et al. 2020). Besides, the weak associations between most individual anthocyanins and isoflavones possibly reflect the tradeoff relationship between their biosynthesis pathways (Wu et al. 2017).

CONCLUSION

In this study, 323 black soybean landraces of Korean origin were cultivated, the contents of five major isoflavones and three major anthocyanins were determined, and the influences of cotyledon color and harvest period on each was assessed. Green cotyledon soybeans had a higher average TIC and TAC than yellow cotyledon soybeans. Besides, landraces harvested in Late-October displayed significantly high average TIC and TAC than landraces harvested in Mid-October and Early-October. In general, cotyledon color appeared to have a significant effect on the TAC, whereas the harvest period appeared to have a significant effect on both the TAC and TIC. Of all the studied landraces, 20 soybeans including S4 (IT:104314), S56 (IT:177573), S70 (IT:177720), S100 (IT:186183), S103 (IT:201834), S113 (IT:201852), S146 (IT:215908), S148 (IT: 216312), S175 (IT:228789), S191 (IT:242611), S192 (IT:252252), S206 (IT: 263106), S218 (IT:263576), S235 (IT:269626), S245 (IT:269847), S256 (IT:274472), S259 (IT:274511), S297 (IT:278716), S309 (IT:283894), and S315 (IT: 285974) had a TAC of > 2000 mg/100 g and a TIC of > 1800 mg/g. Our findings suggest that these landraces could be important sources of high dietary isoflavone and anthocyanin concentrations. Besides, those landraces harvested in Late-October could be targeted to advance their cultivation in Korea.

Supplementary Materials
pbb-9-1-65-supple.pdf
ACKNOWLEDGEMENTS

This work was supported by the Research Program for Agricultural Science & Technology Development (Project No. PJ 014172032021) of the National Institute of Agricultural Sciences, Rural Development Administration (Jeonju, Republic of Korea).

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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