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Metabolism variation and better storability of dark- versus light-coloured soybean (Glycine max L. Merr.) seeds
Food Chemistry 223 (2017) 104–113
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Metabolism variation and better storability of dark- versus light-coloured soybean (Glycine max L. Merr.) seeds Jiang Liu a,b,1,⇑, Wen-ting Qin a,1, Hai-jun Wu a,1, Cai-qiong Yang a, Jun-cai Deng a, Nasir Iqbal a, Wei-guo Liu a,b, Jun-bo Du a,b, Kai Shu a,b, Feng Yang a, Xiao-chun Wang a, Tai-wen Yong a, Wen-yu Yang a,⇑ a b
Key Laboratory of Crop Ecophysiology and Farming System in Southwest, Ministry of Agriculture, Chengdu 611130, China Institute of Ecological Agriculture, Sichuan Agricultural University, Chengdu 611130, China
a r t i c l e
i n f o
Article history: Received 11 August 2016 Received in revised form 8 November 2016 Accepted 12 December 2016 Available online 18 December 2016 Keywords: Coat colour Isoflavone Fatty acid Soybean Storage
a b s t r a c t The effects of storage duration on the seed germination and metabolite profiling of soybean seeds with five different coloured coats were studied. Their germination, constituents and transcript expressions of isoflavones and free fatty acids (FFAs) were compared using chromatographic metabolomic profiling and transcriptome sequencing. The seed water content was characterized using nuclear magnetic resonance (NMR) relaxometry. Results showed that dark-coloured seeds were less inactivated than lightcoloured seeds. The aglycone and b-glucoside concentrations of upstream constituents increased significantly, whereas the acetylglucosides and malonylglucosides of downstream constituents decreased with an increase in the storage period. FFAs increased considerably in the soybean seeds as a result of storage. These results indicate that dark-coloured soybean seeds have better storability than light-coloured seeds, and seed water content plays a role in seed inactivation. It was concluded that there are certain metabolic regularities that are associated with different coloured seed coats of soybeans under storage conditions. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Soybean is an important source of healthy food, and its high global consumption is due to its nutritional and functional bioactive substances, including proteins, fatty acids, and isoflavones (Kim, Jung, Ahn, & Chung, 2005; Ma et al., 2015; Wang, Harp, Hammond, Burrisa, & Fehr, 2001). In general, soybean cultivars have yellow, black, green, brown or mottled seed coats. Most soybean cultivars have a yellow-coloured seed coat, while darkcoloured soybeans are also becoming popular worldwide due to their potentially high functional characteristics (Kumar Dixit, Kumar, Rani, Manjaya, & Bhatnagar, 2011). The physiological and chemical characteristics of soybeans with different coloured seed coats vary. Dark-coloured soybean seeds generally contain high concentrations of anthocyanins or other phenolics that contribute to their high stress resistance (Wu et al., 2013; Zhang, Kawabata, Kitano, & Ashida, 2013). The coats of dark-coloured soybean seeds are harder than those of light-coloured seeds. In particular, the multiple layers of hard seed coat structures and the presence of ⇑ Corresponding authors at: Key Laboratory of Crop Ecophysiology and Farming System in Southwest, Ministry of Agriculture, Chengdu 611130, China (J. Liu). E-mail addresses: [email protected] (J. Liu), [email protected] (W.-y. Yang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.foodchem.2016.12.036 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
chemicals in a continuous layer of cells in the seed coat of black soybeans form a barrier against the permeation of water and oxygen (Zhou, Sekizaki, Yang, Sawa, & Pan, 2010). In agricultural practice, many crop seeds with a high oil concentration deteriorate easily, especially soybean seed. Poor seed storability/longevity leads to poor germination and vigour, and this is an irreversible physiological phenomenon. Storage time and conditions have a considerable influence on grain nutrients and seed quality (De Alencar, Faroni, de Lacerda Filho, Ferreira, & Meneghitti, 2006; Midorikawa et al., 2014; Xiao, Mak, Koch, & Moore, 2013). Almost all chemical characteristics of soybean seeds change during storage. The moisture content, fat content, watersoluble nitrogen content, nitrogen solubility index, sugar and pigment contents, and lipoxygenase activity of seeds have been reported to decrease during storage, whereas the non-protein nitrogen and free fatty acid (FFA) concentrations and the peroxide value have been reported to increase (Narayan, 1988). The abovementioned quality changes in soybeans during storage indirectly affect the sensory qualities of the soy products (Narayan, Chauhan, & Verma, 1988). Although seed aging is a complicated subject, numerous studies have discussed the mechanisms of seed aging and deterioration (Zhang, 1994), including the physiological, biochemical and molecular mechanisms (Liu, Gui, Gao, Ma, & Wang, 2016a). Lipid peroxidation, genetic damage, and integrity loss of cell membranes
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have been investigated in the age-associated physiological processes that occur during storage. Peroxidation and hydrolysis of membrane lipids are usually recognized as essential factors for cellular structural damage caused during seed aging (Wang, Wang, Jing, & Zhang, 2012). Murthy, Kumar, and Sun (2003) studied Vigna radiata seed aging during storage and reported that the loss of seed viability is associated with Maillard reactions, and the contribution of primary biochemical reactions varies under different storage conditions. Undoubtedly, storage results in seed inactivation and metabolic adjustment. However, reports on the effects of storage on soybeans with different coloured seed coats are limited. The goals of the current study were to examine different coloured soybean varieties to determine whether seed coat colour might be associated with storability and to reveal the potential underlying mechanisms, i.e., metabolic physiology and transcriptome levels. 2. Materials and methods 2.1. Plant materials and experimental design Five different seed coat colour genotypes of soybean varieties were tested in the current research. The proto-cultivars were obtained from Clasia, Inc. (Japan) and were labeled as JP-2 (green), JP-6 (black), JP-7 (bicoloured), JP-8 (brown), and JP-16 (yellow). Soybean seeds were harvested from each replicate of each cultivar in each cropping year. From 2012 to 2014, after the harvested seeds were naturally air-dried, all seeds were stored in a controlled low-temperature and humidity storage cabinet (60% relative humidity, 10 °C) for 1–3 years. Laboratory analyses were carried out in 2015; these analyses included a germination test, chromatographic analyses of isoflavones and fatty acids, and NMR and MR imaging analyses of the water content of stored seeds. 2.2. Germination test This experiment was carried out at the Institute of Ecological Agriculture, Sichuan Agricultural University, Chengdu, China, from June to September 2015. Seeds were germinated in 9-cm Petri dishes on two layers of filter paper. A total of 20 seeds were placed on each filter paper, and 10 ml of distilled water was added. During the experiment, constant monitoring ensured sufficient moisture for germination. The seeds were incubated in a 25 °C light incubator with a 12 h day and night alternating condition. Germination was considered to have occurred when radicles (1 mm long) were visible. The germination percentage was recorded every 24 h for 7 days. The experiment was conducted based on a completely randomized design in triplicate (Ling et al., 2014). The following calculations were performed:
Germination rate ð%Þ ¼ ðNumber of seeds germinated in 7 d= total number of seedsÞ 100%: Germination potential ð%Þ ¼ ðNumber of seeds germinated in 3 d= total number of seedsÞ 100%:
Germination index ¼
X Gt ; where Gt represents the number Dt
of germinated seeds on day t; and Dt represents germination days: 2.3. Chromatographic analyses The stored soybean seeds were ground immediately after freeze-drying, then weighed and stored in tightly capped vials
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at 80 °C until analysis. Isoflavone and fatty acid analyses were conducted based on previously published methods (Liu et al., 2016b). Briefly, isoflavone quantification was performed using an HPLC system (Agilent 1100-series) equipped with an ultraviolet (UV) spectroscopy detector connected to a reversephase column. GC–MS analyses of fatty acids were conducted using a GC–MS spectrometer (SHIMADZU QP-2010 system) that was operated in the selected ion-monitoring (SIM) mode. The calibration curve, used as a quantitative approach, was constructed by plotting the peak area ratios of standard concentrations.
2.4. Transcriptome comparison The black (JP-6) and yellow (JP-16) soybeans were compared based on their transcriptome levels. These soybean seeds with different storability were stored for 1–3 years. The soybean seeds were ground into a fine powder for RNA extraction that was quantified as previously described (Gong et al., 2014). A 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to determine RNA quality according to the manufacturer’s protocol. The mRNA-Seq Sample Preparation KitTM (Illumina, San Diego, CA, USA) was used to construct cDNA libraries, and the Agilent Bioanalyzer was used to determine the DNA yield and fragment insert size distribution of each library. The cDNA library products were then sequenced on an Illumina HiSeq2500 sequencing instrument using read lengths of 100 bp.
2.5. NMR relaxometry and MR imaging NMR and MR imaging analyses conducted on stored seeds were performed as described previously by Li et al. (2015). All magnetic resonance experiments reported here were carried out using an 18.17 MHz NMR Analyzer NMI 20 (Niumag Co., Ltd., Shanghai, China) equipped with a standard microimaging birdcage probehead. Approximately 4 g of soybeans were placed in a 25 mm NMR glass tube. The relaxometry measurements were conducted at 32 °C. The typical pulse parameters were as follows: FOVRead = 40 mm, FOVPhase = 40 mm, TR = 20 ms, NS = 8. The TD-NMR relaxation curve was fitted to a multiexponential curve. The original data were transformed into 2D, 196 194 pixel images by Fourier transformation using NMI 20 imaging software (Niumag Electric Corporation, Shanghai, China). Three images of each seed were acquired in a perpendicular direction. The signal intensity of water protons was expressed on a relative basis using the colour scale shown beside the images. In the pseudo-colour images, colours from blue through bright yellow to red represented increasing moisture content in the tissues.
2.6. Statistical analyses All tests were conducted in nonuplicate, the results are reported as the mean ± standard deviation. Variance analyses were performed using the general linear model procedure in SPSS (version 20.0; SPSS, Chicago, USA). Duncan’s multi-range test was used when the samples exhibited significantly different metabolite concentrations at the p < 0.05 level of significance. The concentrations of the different forms of isoflavones and fatty acids in the soybean seeds and the other parameters were subjected to multivariate statistical analysis using MetaboAnalyst 3.0: http://www.metaboanalyst.ca/ (Xia, Sinelnikov, Han, & Wishart, 2015).
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3. Results 3.1. Seed germination The effects of storage on the germination of soybeans with different coloured seed coats are shown in Table S1 and Fig. 1. The results indicate that the germination of all soybean seeds decreased with increasing storage duration (Fig. 1). The germination rate (GR), germination potential (GP), and germination index
(GI) of soybean seeds stored for 1–2 years were 55.0–100.0%, 15.0–98.3%, and 6.8–27.1, respectively. As the storage time extended to three years, the germination parameters decreased considerably, i.e., the GR, GP and GI values decreased to 0.0– 71.7%, 0.0–56.7%, and 0.0–13.7, respectively. The effect of storage duration on soybean seed germination also varied with seed coat colour (Table S1, Fig. 1). The GR values of all soybean seeds stored for 1–2 years (harvested in 2014 and 2013) reached at least 55%. After three years, their germination
Fig. 1. Effects of storage duration on the germination of soybean seed. Quantitative analysis of germination rates of the black variety JP-6 (a), bicoloured variety JP-7 (b), brown variety JP-8 (c), green variety JP-2 (d), yellow variety JP-16 (e), and the variation tendency of germination parameters among different coloured soybean seeds (f). Data presented are the means ± SE (n = 3 biological replicates). GR: germination rate; GP: germination potential; GI: germination index. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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parameters decreased, especially those of the light-coloured soybean seeds. For example, the GR and GP values of the JP-2 (green-coloured seeds, Fig. 1D) and JP-16 (yellow-coloured seeds, Fig. 1E) varieties decreased below 5%, and their GI values decreased below 1.3 after 3 years of storage, which implies that the seeds were almost completely inactivated. The germination parameters of the dark-coloured soybean seeds, i.e. JP-6 (Fig. 1A), JP-7 (Fig. 1B), and JP-8 (Fig. 1C), decreased gradually over time. Although the black-coloured soybean seeds were stored for a longer period (harvested in 2013), these seeds had higher germination parameters compared to the lightcoloured soybean seeds, which were only stored for one year (harvested in 2014). As shown in Fig. 1F, the germination parameters increased with the darkening of the seed coat colour (JP-16 (yellow) ? JP-2 (green) ? JP-8 (brown) ? JP-7 (bicoloured) ? JP-6 (black)) for soybeans that were stored for the same period of time, i.e., three years (harvested in 2012). For two typical varieties, i.e., JP-6 (black) and JP-16 (yellow), large differences were observed in the germination parameters after three years of storage. The germination parameters of the yellow-coloured soybean variety JP-16 had values of 0, which indicated complete inactivation. By contrast, the high GR, GP, and GI values of the black-coloured soybean variety JP-6 were retained, i.e., 65.00%, 56.67%, and 13.74, respectively.
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are shown in Fig. 3 for the different coloured soybean seeds stored over different time periods. The fatty acid constituents (Fig. 3A–E) and their total concentration (Fig. 3F) were consistently maintained at relatively low levels when stored for 1 or 2 years (i.e., PA, 4.06–10.10 mgg1; SA, 1.93– 3.21 mgg1; ALA, 4.09–13.69 mgg1; OA, 10.60–22.32 mgg1; LA, 26.08–40.87 mgg1). After three years of storage, the FFA concentrations increased significantly compared with their corresponding samples stored for 1–2 years (i.e., PA, 9.07–27.42 mgg1; SA, 2.92– 8.25 mgg1; ALA, 10.67–30.43 mgg1; OA, 27.37–51.02 mgg1; LA, 43.16–103.84 mgg1). Additionally, the total FFA concentration increased from 52.19–90.19 mgg1 to 93.19–205.59 mgg1, i.e., twofold higher than the values observed after 1–2 years of storage (Fig. 3F). Further visual analysis indicated that the FFA concentrations of soybean seed stored for 3 years varied with seed coat colour, as shown in Fig. 3. Higher concentrations of fatty acids were detected in the light-coloured soybean seeds, especially PA (i.e., JP-16, 27.42 mgg1) and LA (i.e., JP-16, 103.84 mgg1), which accounted for more than half of the total FFA concentration (Fig. 3A and E). Although the FFA concentrations of the bicolour taupe variety JP7 were higher than the other varieties, it was clear that the lighter-coloured soybean seed contained more FFAs after three years of storage.
3.2. Isoflavone profiling Changes in the isoflavone profiles of the different soybean cultivars are presented in Fig. 2 in response to differences in storage duration. The composition and concentrations of different soybean isoflavones and the biosynthetic pathway are shown. The results showed that the isoflavone concentrations varied with seed coat colour; relatively higher concentrations were detected in the majority of the light-coloured soybean seeds. In particular, malonylglucoside accounted for more than 50% of the total soyisoflavone concentration (Fig. 2C). After three years of storage, the concentrations of various groups of soy-isoflavones exhibited variation patterns that were similar to the corresponding samples that were stored for a shorter period of time, i.e., 1–2 years. However, the bicoloured taupe variety JP-7 was an exception. This variety had a relatively dark colour (Fig. 1B) and although its isoflavone composition and concentration were similar to the lightest colour variety JP-16, it exhibited the highest total isoflavone (T-Iso) concentration (Fig. 2E). The concentrations of different types of isoflavones also varied with storage duration. As the storage duration increased, the concentrations of glucoside and aglycon increased (Fig. 2A and B), whereas those of acetylglucoside and malonylglucoside decreased after storage for 3 years (Fig. 2C and D). The change rates of the TIso concentrations in different coloured seeds were also analyzed. As shown in Fig. 2E, the total concentrations of soy-isoflavones decreased after storage for three years and as the seed coat colour darkened. Different storage durations resulted in significant differences, i.e., in the varieties JP-6, JP-7, JP-8, and JP-16 (Fig. 2E). Although lower T-Iso concentrations were detected in the darkcoloured soybean seeds such as JP-6, its reduction ratio was the highest. The T-Iso concentrations of the yellow variety JP-16 decreased by 4.38% after three years of storage, whereas the TIso reduction ratio reached 16.52% in the black variety JP-6. 3.3. Free fatty acid composition Five major fatty acids, including two saturated fatty acids, namely palmitic acid (PA) and stearic acid (SA), and three major unsaturated fatty acids, namely linoleic acid (LA), oleic acid (OA) and a-linolenic acid (ALA), were detected. These free fatty acids (FFAs), expressed as milligrams per gram of sample dry weight,
3.4. Transcriptome analysis To further determine the metabolic mechanism of the soybean seed response to storage, two typical varieties, JP-6 (black coat) and JP-16 (yellow coat), were selected for transcriptome analysis. Transcription of isoflavonoid and fatty acid genes was quantified by FPKM (fragments per kilobase of transcript per million mapped reads) using the RNA-Seq data set. The relative expression levels of key unigenes involved in isoflavonoid and fatty acid biosynthesis of the two soybean cultivars stored over different periods are shown in Fig. 4. On the whole, the transcription levels of early isoflavonoid pathway genes (PAL, CHS, CHI, and IFS) were much higher than those of late isoflavonoid pathway genes (UGT, AT, and MT). Further comparison of the digital expression profiles of these cultivars revealed a different expression pattern. The transcription levels of key unigenes involved in isoflavonoid biosynthesis were higher in the yellow variety JP-16 than in the black variety JP-6 (Fig. 4A, B). Interestingly, in JP-16, all of the isoflavonoid biosynthesis unigenes were up-regulated during the three years of storage but down-regulated in JP-6. With respect to the digital expression profiles of the fatty acids, the transcription levels of early fatty acid pathway genes (ACP, ACC, FAS, and KCS) were much lower than those of late fatty acid pathway genes (LPS, FAD, and LOX). In particular, LOX, the main catabolic enzyme of fatty acids, exhibited the highest expression levels in all tested samples. The key unigenes involved in fatty acid biosynthesis can be divided into two groups, i.e., synthetases, such as ACP, ACC, FAS, and KCS, and hydrolases, such as LPS, FAD, and LOX. Most of the transcription levels of key unigenes involved in the fatty acid synthetases (ACP, FAS, and KCS) were lower in the yellow variety, JP-16, than in the black variety, JP-6 (Fig. 4C and D). By contrast, the transcription levels of key unigenes involved in the fatty acid hydrolases were remarkably higher in the yellow variety, JP-16, than in the black variety, JP-6 (Fig. 4C and D). Additionally, as shown in Fig. 4C and D, the key unigenes involved in the fatty acid hydrolases (LPS, FAD, and LOX) in JP-16 were significantly upregulated during the three years of storage, whereas the other synthetase genes in JP-16 and all monitored genes in JP-6 did not exhibit obvious changes. In JP-6, LOX was down-regulated during the three years of storage.
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Fig. 2. Soy-isoflavonoid metabolism response to storage duration. Accumulation of aglycone (a), b-glucoside (b), malonyglucoside (c), acetylglucoside (d), and the total isoflavone content (e) in stored soybean seeds. Data presented are the means ± SE (n = 3 biological replicates). Significant differences between storage years were based on a paired t-test: ⁄, P < 0.05, ⁄⁄, P < 0.01. PAL: phenylalanine ammonialyase; CHS: chalcone synthase; CHI: chalcone isomerase; IFS: 2-hydroxyisoflavanone synthase; UGT: glycosyl-transferase; MT: malonyl-transferase; AT: acetyl-transferase.
3.5. Water content and distribution The different coloured soybean seeds, which were stored over various time periods, were subjected to time domain nuclear magnetic resonance (TD-NMR) relaxometry and magnetic resonance imaging (MRI). Based on NMR relaxometry, MRI provides a method to monitor the spatial penetration of water in the soybean seeds.
As shown in Fig. 5, the NMR colour-coded images indicated that the cotyledon was a high moisture area; this area was coloured bright yellow to red. Combined analysis with the detailed seed water content (WC) data in Table S2 clearly indicated that in the soybeans with lighter-coloured seed coats, the WCs increased from 8.71% to 17.41% for the seeds harvested in 2012 and from 9.03% to 18.04% for the seeds harvested in 2013 or 2014. The WC of the
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Fig. 3. Effects of storage duration on the free fatty acids in soybean seed. Accumulation of palmitic acid (a), stearic acid (b), a-linolenic acid (c), oleic acid (d), linoleic acid (e) and total fatty acid (f) in stored soybean seeds. Data presented are the means ± SE (n = 3 biological replicates). Significant differences between storage duration were determined using a paired t-test: ⁄⁄, P < 0.01.
darker-coloured soybean seeds decreased significantly with storage duration, i.e., the black (JP-6), bicoloured (JP-7), and brown (JP-8) varieties. However, the WC of the light-coloured soybean varieties JP-2 and JP-16 did not exhibit an obvious change. For example, the WC of the yellow variety JP-16 only decreased from 18.04% to 17.41%, whereas that of the black variety JP-6 decreased from 15.97% to 9.71%. This information demonstrated that soybean seeds with dark-coloured seed coats lost water more easily than those with light-coloured seed coats. 3.6. Correlation and cluster analyses To further determine the relationships among seed viability, water content and metabolite concentrations, in-depth correla-
tion and cluster analyses were conducted based on the parameters of the different coloured soybean seeds that were stored for three years. As shown in Fig. 6A, all germination parameters (GR, GP and GI) were significantly and negatively correlated with the seed water content, isoflavones and fatty acid constituents. The water content in the seed was significantly positively correlated with the FFA constituents, especially saturated fatty acids (LA and PA). Pearson’s correlation analyses and hierarchical clustering analysis revealed the detailed relationships between the concentrations of the four groups of isoflavones and the five FFAs in the soybean seed. The results indicated strong correlations between the metabolites that participate in closely related pathways, which is consistent with a previous study (Kim et al., 2014).
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Fig. 4. Relative expression of genes involved in the isoflavone and fatty acid pathways of black and yellow soybean seed. Transcription of isoflavonoid and fatty acid genes was quantified using FPKM values. Expression levels of key genes involved in isoflavone metabolism in (a) the yellow soybean variety JP-16 and (b) the black soybean variety JP-6, and key genes involved in fatty acid metabolism in (c) the yellow soybean variety JP-16 and (d) the black soybean variety JP-6. ACP: acyl carrier protein synthase; FAS: fatty acid-CoA synthase; KCS: b-ketoacyl-CoA synthase; ACC: acetyl-CoA carboxylase; Lox: lipoxygenase; FAD: fatty acid desaturase; LPS: lipase. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. NMR colour-coded images of various coloured soybean seeds stored over different time periods. The ‘‘bright part” (high proton density) is coloured red, and the ‘‘dark part” is coloured blue. The colour scale shown beside the images indicates the relative signal intensity of the water protons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The cluster analysis results for the different coloured soybean seeds, based on the above-mentioned parameters, are shown in Fig. 6B. This heat map provides intuitive visualization of the sample information. The five soybean varieties stored for three years can be divided into three groups, i.e., high storability (JP-6), medium storability (JP-7, JP-8, JP-2), and low storability (JP-16). It can be observed that the darker the soybean seed coat colour, the
higher the storability, which is consistent with the results presented above. In addition, the water contents of stored seeds decreased with the darkening of the coat colour; this comparison is presented in Fig. 6B. Most of the tested samples contained high concentrations of FFAs, except the black soybean variety JP-6. The isoflavone concentrations in JP-6 and JP-8 were relatively low compared with the other varieties.
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Fig. 6. Multivariate statistical analysis of the metabolic characteristics associated with the storability of different soybean varieties. Heat map of correlation analysis between germination, water content, and isoflavone and fatty acid concentrations in various soybean seeds (a). Heat map of clustering analysis of metabolic characteristics of various soybean seeds (b). The colour scale shown beside the images indicates the relative signal intensity of the correlations. GR: germination rate; GP: germination potential; GI: germination index; WC: water content; OA: palmitic acid; SA: stearic acid; ALA: a-linolenic acid; OA: oleic acid; LA: linoleic acid; E: aglycones; G: b-glucosides; M: malonyglucosides; A: acetylglucosides.
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4. Discussion 4.1. Dark black soybean seeds were less inactivated Although seed aging is an irreversible physiological phenomenon, many studies have aimed to retard this deteriorative process. However, even if seeds are stored under extremely clean, dry and low temperature conditions, it is difficult to completely prevent their inactivation (Murthy et al., 2003). As a typical legume plant with a high oil content, soybean seed deteriorates easily, leading to necessary seed production and multiplication every 1– 2 years in agricultural practice. Generally, soybean germplasms with dark-coloured seed coats are more similar to the natural wild type, especially black soybean seeds, which present a ‘‘hard seed” characteristic. On the one hand, the hard seed characteristic is a type of seed dormancy; the dormancy of these hard seeds must be broken before sowing (Sun et al., 2014). Consequently, a hard seed is an undesirable characteristic for most growers. On the other hand, as an adaptive characteristic for adverse environments, a hard seed protects soybean seed against deterioration, maintaining high seed vigour over lengthy storage durations (Zhou et al., 2010). The current research indicated that although the storage treatment caused irreversible inactivation of the soybean seeds, the light-coloured seeds deteriorated more easily, whereas the dark-coloured soybean seeds maintained higher seed viability throughout the long-term storage. Dark black soybean seeds were less inactivated during storage; a possible reason may be the hardness of the seed. This characteristic includes both the physical structure and the chemical constituents, i.e., multiple hard layers and phenolics in the dark-coloured seed coat, which effectively prevent external injuries, such as oxidization and insect damage. 4.2. Storage had a strong influence on the metabolism variation As the main functional constituents of soybean seed, isoflavones and fatty acids not only represent various bioactivities in the medicinal field but also play key roles in plant physiological resistance. This study indicated that glucoside and aglycon increased with storage duration, whereas acetylglucoside and malonylglucoside decreased as a result of storage. This is consistent with previous studies (Kim, Kim, Hahn, & Chung, 2005; Lee et al., 2003), but the mechanisms involved in soy isoflavone metabolism are not well understood. Further comparative transcriptome analysis between two typical soybean seeds with different storability was conducted. The results demonstrated that isoflavone metabolism in the soybean seed did not stop during storage. The isoflavone metabolism in the light-coloured soybean seed was more susceptible to the environment, whereas the dark-coloured soybean seed appeared to be relatively stable. The expression levels of key upstream genes (PAL, CHS, CHI, and IFS) involved in isoflavone metabolism were significantly higher than those of downstream genes (UGT, AT, and MT), resulting in the transformation, and conversion, of different types of isoflavones in soybean seed being affected by storage. Furthermore, the high instability of malonyl derivatives of isoflavonoids also caused their conversion to corresponding glycosyl conjugates. Malonyl-conjugates followed by glycosyl-conjugates are the main forms of isoflavones in soybean seed, which account for more than 80% of the total content (Liu et al., 2016b). The conversion between glucoside and malonylglucoside represents the main transformation involved in isoflavone metabolism in soybean seed. The mechanism through which plant vacuoles confer stability to malonyl isoflavone conjugates remains unknown (Dastmalchi & Dhaubhadel, 2014). However, the following occurs in the conversion of malonylglucoside to glucoside: dehydration of the soybean seed during storage results in vacuolar atrophy, leading to isofla-
vone re-compartmentalization, which induces isoflavone transport from the central vacuole of the cell to the apoplast. Aglycon and glucoside are more involved in the defense interaction with the environment, compared with their corresponding malonyl or acetyl derivatives (Jones & Vogt, 2001). For example, the antioxidant activities of aglycons have been reported to be higher than those of corresponding glucoside derivatives (Mora, Payá, Ríos, & Alcaraz, 1990). This study indicated that the degree of conversion from malonylglucoside to glucoside in dark-coloured soybean seed was higher than in light-coloured seed during storage. This information implies that dark-coloured soybean seed may have a greater resistance potential based on isoflavone conversion. Storage also had a strong influence on the metabolism of fatty acids. Lipoxygenase plays a key role in seed aging (Devaiah et al., 2007). High amounts of FFAs imply the formation of peroxides and hydrolytic changes in fat components (Narayan, 1988). Compared with the up-regulation of LOX in yellow soybean seed, LOX in black soybean seed was down-regulated during the three years of storage. Significantly higher increases in FFAs were also detected in light-coloured soybean seed compared with the dark-coloured samples. The higher expression levels of late fatty acid pathway genes, especially LOX in light-coloured soybean seed, may account for the higher levels of FFAs as a result of storage. This also implies that dark-coloured soybean seed may have a greater resistance potential based on the low levels of FFAs, which indicate damage caused by peroxidation and hydrolysis (Devaiah et al., 2007). 4.3. Water content accelerates seed inactivation Several scientific hypotheses related to seed deterioration have been proposed in previous studies. A well-known theory is that free radical assault on mitochondrial membranes can be involved. This theory suggests that active oxygen species (AOS), which are free radicals generated during the oxidizing reaction in the interior of the seed, attack intracellular lipids and their constituent proteins, damaging the integrity of the plasma membrane and resulting in seed deterioration (Sanz, Pamplona, & Barja, 2006). However, cells are endowed with detoxifying enzymes and antioxidant compounds that scavenge AOS and participate in seed survival (Bailly, 2004). McDonald (1999) demonstrated that long-term storage correlates with higher accumulated AOS levels, and membrane lipid autoxidation is the main source of the accumulated AOS. Dry seed tissue metabolism can shut down due to a reduction in the mobility of molecules, which was controlled by a glass matrix structure (Bailly, 2004). Mira, González-Benito, Hill, and Walters (2010) studied the production rate of volatile peroxidation by-products of lettuce seeds during storage. These authors demonstrated that the deterioration mechanisms involved in viability loss differed depending on the seed water content; a low water content can retard seed deterioration. In this study, TD-NMR analysis indicated that the dark-coloured seed lost water more easily than the lightcoloured seed during long-term storage. This implies that darkcoloured seed has a greater resistance potential based on the high rate of water loss, which decreases AOS to mitigate oxidation. The multiple hard layers of the seed coat of dark-coloured soybean, especially those of black soybean, lead to resistance to water absorption, thus causing seed dormancy (Mullin & Xu, 2001). However, our study indicated that the hard seed of black soybean facilitated water loss and resulted in higher germination parameters. This seemingly contradictory finding suggests a two-way regulatory mechanism acts on the surface of soybean seed. Research has indicated that there are two layers of palisade cells in the soybean seed coat, one of which is located in the outer epidermal layer, referred to as the ‘‘reverse palisade layer” (Sun et al., 2014). This ‘‘reverse palisade layer” acts as a valve structure to control water circulation. For example, this layer only allows water
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dispersion, preventing the entry of water from the outside environment. This type of regulating function is especially evident in black soybean varieties. 5. Conclusions In conclusion, comparative analysis of seed vitality, metabolism and water content between dark- and light-coloured soybean seeds subjected to different storage durations revealed the partial mechanisms of seed deterioration, including the conversion of different types of isoflavones that function as infiltration regulators (Yumei & Zhou, 2011); the release of FFAs, which function as markers of lipid membrane oxidation; and the water-holding capacity of the seed vacuole, which functions as an oxidation accelerator. A combination of these factors resulted in greater inactivation of light-coloured soybean seeds, whereas dark-coloured varieties had better storability. Comparative transcriptomic and NMR relaxometry analyses revealed that the above-mentioned mechanisms may be mediated by lipoxygenase (IFS) and 2-hydroxyisoflavanone synthase (LOX) genes, and the activities of these enzymes may be regulated by the cotyledon water content. Additionally, there was a certain regularity in the storability and metabolic physiology of the different coloured soybean seeds, especially the pure coloured seed (e.g., JP-6, JP-8, JP-2, JP-16). The in-depth correlation and cluster analyses confirmed this finding. However, inconsistent results were observed for the bicoloured soybean seed (i.e., JP-7) that was checkered with black and white, which was similar to the taupe seed. The physiological and metabolic parameters of the bicoloured soybean seed (JP-7) had values that fell between the values of black (JP-6) soybean seed and brown (JP-8) soybean seed. For example, the seed vigour of JP-7 was higher than that of JP-8 but lower than that of JP-6. These results suggest that seed coat pigment is closely related to seed storability. However, determination of a quantitative relationship between pigment content and seed vitality requires further study. Consequently, further investigation of the effects of soybean seed colour and quantitative analysis of seed coat pigments should be conducted. Acknowledgements This study was financially supported by the National Natural Science Foundation of China (Grant No. 31401329) and the China Postdoctoral Science Foundation (Grant No. 2014M560724). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2016. 12.030. References Bailly, C. (2004). Active oxygen species and antioxidants in seed biology. Seed Science Research, 14, 93–107. Dastmalchi, M., & Dhaubhadel, S. (2014). Soybean seed isoflavonoids: Biosynthesis and regulation. In R. Jetter (Ed.). Phytochemicals – biosynthesis, function and application (Vol. 44, pp. 1–21). Heidelberg: Springer. De Alencar, E. R., Faroni, L. R. D. A., de Lacerda Filho, A. F., Ferreira, L. G., & Meneghitti, M. R. (2006). Influence of different storage conditions on soybean grain quality. In: Proceedings of the 9th international working conference on stored-product protection (pp. 30–37). Devaiah, S. P., Pan, X., Hong, Y., Roth, M., Welti, R., & Wang, X. (2007). Enhancing seed quality and viability by suppressing phospholipase D in Arabidopsis. Plant Journal, 50, 950–957. Gong, W., Qi, P., Du, J., Sun, X., Wu, X., Song, C., & Yang, W. (2014). Transcriptome analysis of shade-induced inhibition on leaf size in relay intercropped soybean. PLoS ONE, 9, e98465. Jones, P., & Vogt, T. (2001). Glycosyltransferases in secondary plant metabolism: tranquilizers and stimulant controllers. Planta, 213, 164–174.
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Kim, S. H., Jung, W. S., Ahn, J. K., & Chung, I. M. (2005). Analysis of isoflavone concentration and composition in soybean [Glycine max (L.)] seeds between the cropping year and storage for 3 years. European Food Research and Technology, 220, 207–214. Kim, J. J., Kim, S. H., Hahn, S. J., & Chung, I. M. (2005). Changing soybean isoflavone composition and concentrations under two different storage conditions over three years. Food Research International, 38, 435–444. Kim, J. K., Kim, E. H., Park, I., Yu, B. R., Lim, J. D., Lee, Y. S., ... Chung, I. M. (2014). Isoflavones profiling of soybean [Glycine max (L.) Merrill] germplasms and their correlations with metabolic pathways. Food Chemistry, 153, 258–264. Kumar Dixit, A., Kumar, V., Rani, A., Manjaya, J. G., & Bhatnagar, D. (2011). Effect of gamma irradiation on lipoxygenases, trypsin inhibitor, raffinose family oligosaccharides and nutritional factors of different seed coat colored soybean (Glycine max L.). Radiation Physics and Chemistry, 80, 597–603. Lee, S. J., Ahn, J. K., Kim, S. H., Kim, J. T., Han, S. J., Jung, M. Y., & Chung, I. M. (2003). Variation in isoflavone of soybean cultivars with location and storage duration. Journal of Agricultural and Food Chemistry, 51, 3382–3389. Li, T., Rui, X., Tu, C., Li, W., Wang, K., Huang, L., & Dong, M. (2015). NMR relaxometry and imaging to study water dynamics during soaking and blanching of soybean. International Journal of Food Engineering, 12, 181–188. Ling, L., Jiafeng, J., Jiangang, L., Minchong, S., Xin, H., Hanliang, S., & Yuanhua, D. (2014). Effects of cold plasma treatment on seed germination and seedling growth of soybean. Scientific Reports, 4, 5859. Liu, J., Gui, J., Gao, W., Ma, J., & Wang, Q. (2016a). Review of the physiological and biochemical reactions and molecular mechanisms of seed aging. Acta Ecologica Sinica, 36. Liu, J., Yang, C.-Q., Zhang, Q., Lou, Y., Wu, H.-J., Deng, J.-C., ... Yang, W.-Y. (2016b). Partial improvements in the flavor quality of soybean seeds using intercropping systems with appropriate shading. Food Chemistry, 207, 107–114. Ma, L., Li, B., Han, F., Yan, S., Wang, L., & Sun, J. (2015). Evaluation of the chemical quality traits of soybean seeds, as related to sensory attributes of soymilk. Food Chemistry, 173, 694–701. McDonald, M. B. (1999). Seed deterioration: Physiology, repair and assessment. Seed Science and Technology, 27, 177–237. Midorikawa, K., Kuroda, M., Terauchi, K., Hoshi, M., Ikenaga, S., Ishimaru, Y., ... Asakura, T. (2014). Additional nitrogen fertilization at heading time of rice down-regulates cellulose synthesis in seed endosperm. PLoS ONE, 9, e98738. Mira, S., González-Benito, M. E., Hill, L. M., & Walters, C. (2010). Characterization of volatile production during storage of lettuce (Lactuca sativa) seed. Journal of Experimental Botany, 61, 3915–3924. Mora, A., Payá, M., Ríos, J. L., & Alcaraz, M. J. (1990). Structure-activity relationships of polymethoxyflavones and other flavonoids as inhibitors of non-enzymic lipid peroxidation. Biochemical Pharmacology, 40, 793–797. Mullin, W. J., & Xu, W. (2001). Study of soybean seed coat components and their relationship to water absorption. Journal of Agricultural and Food Chemistry, 49, 5331–5335. Murthy, U. M. N., Kumar, P. P., & Sun, W. Q. (2003). Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: Lipid peroxidation, sugar hydrolysis, Maillard reactions and their relationship to glass state transition. Journal of Experimental Botany, 54, 1057–1067. Narayan, R. (1988). Changes in the quality of soybean during storage. Part 1—Effect of storage on some physico-chemical properties of soybean. Food Chemistry, 27, 13–23. Narayan, R., Chauhan, G., & Verma, N. (1988). Changes in the quality of soybean during storage. Part 2—Effect of soybean storage on the sensory qualities of the products made there from. Food Chemistry, 30, 181–190. Sanz, A., Pamplona, R., & Barja, G. (2006). Is the mitochondrial free radical theory of aging intact? Antioxidants & Redox Signaling, 8, 582–599. Sun, X., Wang, Z., Li, S., Men, F., Wang, S., & Zhang, J. (2014). Progress on formation mechanism and breaking methods of hard seed in soybean. Soybean Science & Technology, 3, 23–27. Wang, T., Harp, T., Hammond, E. G., Burrisa, J. S., & Fehr, W. R. (2001). Seed physiological performance of soybeans with altered saturated fatty acid contents. Seed Science Research, 11, 93–97. Wang, F., Wang, R., Jing, W., & Zhang, W. (2012). Quantitative dissection of lipid degradation in rice seeds during accelerated aging. Plant Growth Regulation, 66, 49–58. Wu, K., Xiao, S., Chen, Q., Wang, Q., Zhang, Y., Li, K., ... Chen, L. (2013). Changes in the activity and transcription of antioxidant enzymes in response to al stress in black soybeans. Plant Molecular Biology Reporter, 31, 141–150. Xia, J., Sinelnikov, I. V., Han, B., & Wishart, D. S. (2015). MetaboAnalyst 3.0—making metabolomics more meaningful. Nucleic Acids Research, 43, W251–W257. Xiao, Z., Mak, A., Koch, K., & Moore, R. B. (2013). A molecular complex of bovine milk protein and oleic acid selectively kills cancer cells in vitro and inhibits tumour growth in an orthotopic rat bladder tumour model. BJU International, 112, E201–E210. Yumei, W. U., & Zhou, Q. (2011). Effects of seed soaking with soybean isoflavones on soybean seedlings under salt stress. Acta Ecologica Sinica, 31, 6669–6676. Zhang, M. (1994). A mechanism of seed deterioration in relation to the volatile compounds evolved by dry seeds themselves. Seed Science Research, 4, 49–56. Zhang, T., Kawabata, K., Kitano, R., & Ashida, H. (2013). Preventive effects of black soybean seed coat polyphenols against DNA damage in Salmonella typhimurium. Food Science and Technology Research, 19, 685–690. Zhou, S., Sekizaki, H., Yang, Z. H., Sawa, S., & Pan, J. (2010). Phenolics in the seed coat of wild soybean (Glycine soja) and their significance for seed hardness and seed germination. Journal of Agricultural and Food Chemistry, 58, 10972–10978.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Metabolism variation and better storability of dark- versus light-coloured soybean (Glycine max L. Merr.) seeds Jiang Liu a,b,1,⇑, Wen-ting Qin a,1, Hai-jun Wu a,1, Cai-qiong Yang a, Jun-cai Deng a, Nasir Iqbal a, Wei-guo Liu a,b, Jun-bo Du a,b, Kai Shu a,b, Feng Yang a, Xiao-chun Wang a, Tai-wen Yong a, Wen-yu Yang a,⇑ a b
Key Laboratory of Crop Ecophysiology and Farming System in Southwest, Ministry of Agriculture, Chengdu 611130, China Institute of Ecological Agriculture, Sichuan Agricultural University, Chengdu 611130, China
a r t i c l e
i n f o
Article history: Received 11 August 2016 Received in revised form 8 November 2016 Accepted 12 December 2016 Available online 18 December 2016 Keywords: Coat colour Isoflavone Fatty acid Soybean Storage
a b s t r a c t The effects of storage duration on the seed germination and metabolite profiling of soybean seeds with five different coloured coats were studied. Their germination, constituents and transcript expressions of isoflavones and free fatty acids (FFAs) were compared using chromatographic metabolomic profiling and transcriptome sequencing. The seed water content was characterized using nuclear magnetic resonance (NMR) relaxometry. Results showed that dark-coloured seeds were less inactivated than lightcoloured seeds. The aglycone and b-glucoside concentrations of upstream constituents increased significantly, whereas the acetylglucosides and malonylglucosides of downstream constituents decreased with an increase in the storage period. FFAs increased considerably in the soybean seeds as a result of storage. These results indicate that dark-coloured soybean seeds have better storability than light-coloured seeds, and seed water content plays a role in seed inactivation. It was concluded that there are certain metabolic regularities that are associated with different coloured seed coats of soybeans under storage conditions. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Soybean is an important source of healthy food, and its high global consumption is due to its nutritional and functional bioactive substances, including proteins, fatty acids, and isoflavones (Kim, Jung, Ahn, & Chung, 2005; Ma et al., 2015; Wang, Harp, Hammond, Burrisa, & Fehr, 2001). In general, soybean cultivars have yellow, black, green, brown or mottled seed coats. Most soybean cultivars have a yellow-coloured seed coat, while darkcoloured soybeans are also becoming popular worldwide due to their potentially high functional characteristics (Kumar Dixit, Kumar, Rani, Manjaya, & Bhatnagar, 2011). The physiological and chemical characteristics of soybeans with different coloured seed coats vary. Dark-coloured soybean seeds generally contain high concentrations of anthocyanins or other phenolics that contribute to their high stress resistance (Wu et al., 2013; Zhang, Kawabata, Kitano, & Ashida, 2013). The coats of dark-coloured soybean seeds are harder than those of light-coloured seeds. In particular, the multiple layers of hard seed coat structures and the presence of ⇑ Corresponding authors at: Key Laboratory of Crop Ecophysiology and Farming System in Southwest, Ministry of Agriculture, Chengdu 611130, China (J. Liu). E-mail addresses: [email protected] (J. Liu), [email protected] (W.-y. Yang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.foodchem.2016.12.036 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
chemicals in a continuous layer of cells in the seed coat of black soybeans form a barrier against the permeation of water and oxygen (Zhou, Sekizaki, Yang, Sawa, & Pan, 2010). In agricultural practice, many crop seeds with a high oil concentration deteriorate easily, especially soybean seed. Poor seed storability/longevity leads to poor germination and vigour, and this is an irreversible physiological phenomenon. Storage time and conditions have a considerable influence on grain nutrients and seed quality (De Alencar, Faroni, de Lacerda Filho, Ferreira, & Meneghitti, 2006; Midorikawa et al., 2014; Xiao, Mak, Koch, & Moore, 2013). Almost all chemical characteristics of soybean seeds change during storage. The moisture content, fat content, watersoluble nitrogen content, nitrogen solubility index, sugar and pigment contents, and lipoxygenase activity of seeds have been reported to decrease during storage, whereas the non-protein nitrogen and free fatty acid (FFA) concentrations and the peroxide value have been reported to increase (Narayan, 1988). The abovementioned quality changes in soybeans during storage indirectly affect the sensory qualities of the soy products (Narayan, Chauhan, & Verma, 1988). Although seed aging is a complicated subject, numerous studies have discussed the mechanisms of seed aging and deterioration (Zhang, 1994), including the physiological, biochemical and molecular mechanisms (Liu, Gui, Gao, Ma, & Wang, 2016a). Lipid peroxidation, genetic damage, and integrity loss of cell membranes
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have been investigated in the age-associated physiological processes that occur during storage. Peroxidation and hydrolysis of membrane lipids are usually recognized as essential factors for cellular structural damage caused during seed aging (Wang, Wang, Jing, & Zhang, 2012). Murthy, Kumar, and Sun (2003) studied Vigna radiata seed aging during storage and reported that the loss of seed viability is associated with Maillard reactions, and the contribution of primary biochemical reactions varies under different storage conditions. Undoubtedly, storage results in seed inactivation and metabolic adjustment. However, reports on the effects of storage on soybeans with different coloured seed coats are limited. The goals of the current study were to examine different coloured soybean varieties to determine whether seed coat colour might be associated with storability and to reveal the potential underlying mechanisms, i.e., metabolic physiology and transcriptome levels. 2. Materials and methods 2.1. Plant materials and experimental design Five different seed coat colour genotypes of soybean varieties were tested in the current research. The proto-cultivars were obtained from Clasia, Inc. (Japan) and were labeled as JP-2 (green), JP-6 (black), JP-7 (bicoloured), JP-8 (brown), and JP-16 (yellow). Soybean seeds were harvested from each replicate of each cultivar in each cropping year. From 2012 to 2014, after the harvested seeds were naturally air-dried, all seeds were stored in a controlled low-temperature and humidity storage cabinet (60% relative humidity, 10 °C) for 1–3 years. Laboratory analyses were carried out in 2015; these analyses included a germination test, chromatographic analyses of isoflavones and fatty acids, and NMR and MR imaging analyses of the water content of stored seeds. 2.2. Germination test This experiment was carried out at the Institute of Ecological Agriculture, Sichuan Agricultural University, Chengdu, China, from June to September 2015. Seeds were germinated in 9-cm Petri dishes on two layers of filter paper. A total of 20 seeds were placed on each filter paper, and 10 ml of distilled water was added. During the experiment, constant monitoring ensured sufficient moisture for germination. The seeds were incubated in a 25 °C light incubator with a 12 h day and night alternating condition. Germination was considered to have occurred when radicles (1 mm long) were visible. The germination percentage was recorded every 24 h for 7 days. The experiment was conducted based on a completely randomized design in triplicate (Ling et al., 2014). The following calculations were performed:
Germination rate ð%Þ ¼ ðNumber of seeds germinated in 7 d= total number of seedsÞ 100%: Germination potential ð%Þ ¼ ðNumber of seeds germinated in 3 d= total number of seedsÞ 100%:
Germination index ¼
X Gt ; where Gt represents the number Dt
of germinated seeds on day t; and Dt represents germination days: 2.3. Chromatographic analyses The stored soybean seeds were ground immediately after freeze-drying, then weighed and stored in tightly capped vials
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at 80 °C until analysis. Isoflavone and fatty acid analyses were conducted based on previously published methods (Liu et al., 2016b). Briefly, isoflavone quantification was performed using an HPLC system (Agilent 1100-series) equipped with an ultraviolet (UV) spectroscopy detector connected to a reversephase column. GC–MS analyses of fatty acids were conducted using a GC–MS spectrometer (SHIMADZU QP-2010 system) that was operated in the selected ion-monitoring (SIM) mode. The calibration curve, used as a quantitative approach, was constructed by plotting the peak area ratios of standard concentrations.
2.4. Transcriptome comparison The black (JP-6) and yellow (JP-16) soybeans were compared based on their transcriptome levels. These soybean seeds with different storability were stored for 1–3 years. The soybean seeds were ground into a fine powder for RNA extraction that was quantified as previously described (Gong et al., 2014). A 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to determine RNA quality according to the manufacturer’s protocol. The mRNA-Seq Sample Preparation KitTM (Illumina, San Diego, CA, USA) was used to construct cDNA libraries, and the Agilent Bioanalyzer was used to determine the DNA yield and fragment insert size distribution of each library. The cDNA library products were then sequenced on an Illumina HiSeq2500 sequencing instrument using read lengths of 100 bp.
2.5. NMR relaxometry and MR imaging NMR and MR imaging analyses conducted on stored seeds were performed as described previously by Li et al. (2015). All magnetic resonance experiments reported here were carried out using an 18.17 MHz NMR Analyzer NMI 20 (Niumag Co., Ltd., Shanghai, China) equipped with a standard microimaging birdcage probehead. Approximately 4 g of soybeans were placed in a 25 mm NMR glass tube. The relaxometry measurements were conducted at 32 °C. The typical pulse parameters were as follows: FOVRead = 40 mm, FOVPhase = 40 mm, TR = 20 ms, NS = 8. The TD-NMR relaxation curve was fitted to a multiexponential curve. The original data were transformed into 2D, 196 194 pixel images by Fourier transformation using NMI 20 imaging software (Niumag Electric Corporation, Shanghai, China). Three images of each seed were acquired in a perpendicular direction. The signal intensity of water protons was expressed on a relative basis using the colour scale shown beside the images. In the pseudo-colour images, colours from blue through bright yellow to red represented increasing moisture content in the tissues.
2.6. Statistical analyses All tests were conducted in nonuplicate, the results are reported as the mean ± standard deviation. Variance analyses were performed using the general linear model procedure in SPSS (version 20.0; SPSS, Chicago, USA). Duncan’s multi-range test was used when the samples exhibited significantly different metabolite concentrations at the p < 0.05 level of significance. The concentrations of the different forms of isoflavones and fatty acids in the soybean seeds and the other parameters were subjected to multivariate statistical analysis using MetaboAnalyst 3.0: http://www.metaboanalyst.ca/ (Xia, Sinelnikov, Han, & Wishart, 2015).
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3. Results 3.1. Seed germination The effects of storage on the germination of soybeans with different coloured seed coats are shown in Table S1 and Fig. 1. The results indicate that the germination of all soybean seeds decreased with increasing storage duration (Fig. 1). The germination rate (GR), germination potential (GP), and germination index
(GI) of soybean seeds stored for 1–2 years were 55.0–100.0%, 15.0–98.3%, and 6.8–27.1, respectively. As the storage time extended to three years, the germination parameters decreased considerably, i.e., the GR, GP and GI values decreased to 0.0– 71.7%, 0.0–56.7%, and 0.0–13.7, respectively. The effect of storage duration on soybean seed germination also varied with seed coat colour (Table S1, Fig. 1). The GR values of all soybean seeds stored for 1–2 years (harvested in 2014 and 2013) reached at least 55%. After three years, their germination
Fig. 1. Effects of storage duration on the germination of soybean seed. Quantitative analysis of germination rates of the black variety JP-6 (a), bicoloured variety JP-7 (b), brown variety JP-8 (c), green variety JP-2 (d), yellow variety JP-16 (e), and the variation tendency of germination parameters among different coloured soybean seeds (f). Data presented are the means ± SE (n = 3 biological replicates). GR: germination rate; GP: germination potential; GI: germination index. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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parameters decreased, especially those of the light-coloured soybean seeds. For example, the GR and GP values of the JP-2 (green-coloured seeds, Fig. 1D) and JP-16 (yellow-coloured seeds, Fig. 1E) varieties decreased below 5%, and their GI values decreased below 1.3 after 3 years of storage, which implies that the seeds were almost completely inactivated. The germination parameters of the dark-coloured soybean seeds, i.e. JP-6 (Fig. 1A), JP-7 (Fig. 1B), and JP-8 (Fig. 1C), decreased gradually over time. Although the black-coloured soybean seeds were stored for a longer period (harvested in 2013), these seeds had higher germination parameters compared to the lightcoloured soybean seeds, which were only stored for one year (harvested in 2014). As shown in Fig. 1F, the germination parameters increased with the darkening of the seed coat colour (JP-16 (yellow) ? JP-2 (green) ? JP-8 (brown) ? JP-7 (bicoloured) ? JP-6 (black)) for soybeans that were stored for the same period of time, i.e., three years (harvested in 2012). For two typical varieties, i.e., JP-6 (black) and JP-16 (yellow), large differences were observed in the germination parameters after three years of storage. The germination parameters of the yellow-coloured soybean variety JP-16 had values of 0, which indicated complete inactivation. By contrast, the high GR, GP, and GI values of the black-coloured soybean variety JP-6 were retained, i.e., 65.00%, 56.67%, and 13.74, respectively.
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are shown in Fig. 3 for the different coloured soybean seeds stored over different time periods. The fatty acid constituents (Fig. 3A–E) and their total concentration (Fig. 3F) were consistently maintained at relatively low levels when stored for 1 or 2 years (i.e., PA, 4.06–10.10 mgg1; SA, 1.93– 3.21 mgg1; ALA, 4.09–13.69 mgg1; OA, 10.60–22.32 mgg1; LA, 26.08–40.87 mgg1). After three years of storage, the FFA concentrations increased significantly compared with their corresponding samples stored for 1–2 years (i.e., PA, 9.07–27.42 mgg1; SA, 2.92– 8.25 mgg1; ALA, 10.67–30.43 mgg1; OA, 27.37–51.02 mgg1; LA, 43.16–103.84 mgg1). Additionally, the total FFA concentration increased from 52.19–90.19 mgg1 to 93.19–205.59 mgg1, i.e., twofold higher than the values observed after 1–2 years of storage (Fig. 3F). Further visual analysis indicated that the FFA concentrations of soybean seed stored for 3 years varied with seed coat colour, as shown in Fig. 3. Higher concentrations of fatty acids were detected in the light-coloured soybean seeds, especially PA (i.e., JP-16, 27.42 mgg1) and LA (i.e., JP-16, 103.84 mgg1), which accounted for more than half of the total FFA concentration (Fig. 3A and E). Although the FFA concentrations of the bicolour taupe variety JP7 were higher than the other varieties, it was clear that the lighter-coloured soybean seed contained more FFAs after three years of storage.
3.2. Isoflavone profiling Changes in the isoflavone profiles of the different soybean cultivars are presented in Fig. 2 in response to differences in storage duration. The composition and concentrations of different soybean isoflavones and the biosynthetic pathway are shown. The results showed that the isoflavone concentrations varied with seed coat colour; relatively higher concentrations were detected in the majority of the light-coloured soybean seeds. In particular, malonylglucoside accounted for more than 50% of the total soyisoflavone concentration (Fig. 2C). After three years of storage, the concentrations of various groups of soy-isoflavones exhibited variation patterns that were similar to the corresponding samples that were stored for a shorter period of time, i.e., 1–2 years. However, the bicoloured taupe variety JP-7 was an exception. This variety had a relatively dark colour (Fig. 1B) and although its isoflavone composition and concentration were similar to the lightest colour variety JP-16, it exhibited the highest total isoflavone (T-Iso) concentration (Fig. 2E). The concentrations of different types of isoflavones also varied with storage duration. As the storage duration increased, the concentrations of glucoside and aglycon increased (Fig. 2A and B), whereas those of acetylglucoside and malonylglucoside decreased after storage for 3 years (Fig. 2C and D). The change rates of the TIso concentrations in different coloured seeds were also analyzed. As shown in Fig. 2E, the total concentrations of soy-isoflavones decreased after storage for three years and as the seed coat colour darkened. Different storage durations resulted in significant differences, i.e., in the varieties JP-6, JP-7, JP-8, and JP-16 (Fig. 2E). Although lower T-Iso concentrations were detected in the darkcoloured soybean seeds such as JP-6, its reduction ratio was the highest. The T-Iso concentrations of the yellow variety JP-16 decreased by 4.38% after three years of storage, whereas the TIso reduction ratio reached 16.52% in the black variety JP-6. 3.3. Free fatty acid composition Five major fatty acids, including two saturated fatty acids, namely palmitic acid (PA) and stearic acid (SA), and three major unsaturated fatty acids, namely linoleic acid (LA), oleic acid (OA) and a-linolenic acid (ALA), were detected. These free fatty acids (FFAs), expressed as milligrams per gram of sample dry weight,
3.4. Transcriptome analysis To further determine the metabolic mechanism of the soybean seed response to storage, two typical varieties, JP-6 (black coat) and JP-16 (yellow coat), were selected for transcriptome analysis. Transcription of isoflavonoid and fatty acid genes was quantified by FPKM (fragments per kilobase of transcript per million mapped reads) using the RNA-Seq data set. The relative expression levels of key unigenes involved in isoflavonoid and fatty acid biosynthesis of the two soybean cultivars stored over different periods are shown in Fig. 4. On the whole, the transcription levels of early isoflavonoid pathway genes (PAL, CHS, CHI, and IFS) were much higher than those of late isoflavonoid pathway genes (UGT, AT, and MT). Further comparison of the digital expression profiles of these cultivars revealed a different expression pattern. The transcription levels of key unigenes involved in isoflavonoid biosynthesis were higher in the yellow variety JP-16 than in the black variety JP-6 (Fig. 4A, B). Interestingly, in JP-16, all of the isoflavonoid biosynthesis unigenes were up-regulated during the three years of storage but down-regulated in JP-6. With respect to the digital expression profiles of the fatty acids, the transcription levels of early fatty acid pathway genes (ACP, ACC, FAS, and KCS) were much lower than those of late fatty acid pathway genes (LPS, FAD, and LOX). In particular, LOX, the main catabolic enzyme of fatty acids, exhibited the highest expression levels in all tested samples. The key unigenes involved in fatty acid biosynthesis can be divided into two groups, i.e., synthetases, such as ACP, ACC, FAS, and KCS, and hydrolases, such as LPS, FAD, and LOX. Most of the transcription levels of key unigenes involved in the fatty acid synthetases (ACP, FAS, and KCS) were lower in the yellow variety, JP-16, than in the black variety, JP-6 (Fig. 4C and D). By contrast, the transcription levels of key unigenes involved in the fatty acid hydrolases were remarkably higher in the yellow variety, JP-16, than in the black variety, JP-6 (Fig. 4C and D). Additionally, as shown in Fig. 4C and D, the key unigenes involved in the fatty acid hydrolases (LPS, FAD, and LOX) in JP-16 were significantly upregulated during the three years of storage, whereas the other synthetase genes in JP-16 and all monitored genes in JP-6 did not exhibit obvious changes. In JP-6, LOX was down-regulated during the three years of storage.
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Fig. 2. Soy-isoflavonoid metabolism response to storage duration. Accumulation of aglycone (a), b-glucoside (b), malonyglucoside (c), acetylglucoside (d), and the total isoflavone content (e) in stored soybean seeds. Data presented are the means ± SE (n = 3 biological replicates). Significant differences between storage years were based on a paired t-test: ⁄, P < 0.05, ⁄⁄, P < 0.01. PAL: phenylalanine ammonialyase; CHS: chalcone synthase; CHI: chalcone isomerase; IFS: 2-hydroxyisoflavanone synthase; UGT: glycosyl-transferase; MT: malonyl-transferase; AT: acetyl-transferase.
3.5. Water content and distribution The different coloured soybean seeds, which were stored over various time periods, were subjected to time domain nuclear magnetic resonance (TD-NMR) relaxometry and magnetic resonance imaging (MRI). Based on NMR relaxometry, MRI provides a method to monitor the spatial penetration of water in the soybean seeds.
As shown in Fig. 5, the NMR colour-coded images indicated that the cotyledon was a high moisture area; this area was coloured bright yellow to red. Combined analysis with the detailed seed water content (WC) data in Table S2 clearly indicated that in the soybeans with lighter-coloured seed coats, the WCs increased from 8.71% to 17.41% for the seeds harvested in 2012 and from 9.03% to 18.04% for the seeds harvested in 2013 or 2014. The WC of the
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Fig. 3. Effects of storage duration on the free fatty acids in soybean seed. Accumulation of palmitic acid (a), stearic acid (b), a-linolenic acid (c), oleic acid (d), linoleic acid (e) and total fatty acid (f) in stored soybean seeds. Data presented are the means ± SE (n = 3 biological replicates). Significant differences between storage duration were determined using a paired t-test: ⁄⁄, P < 0.01.
darker-coloured soybean seeds decreased significantly with storage duration, i.e., the black (JP-6), bicoloured (JP-7), and brown (JP-8) varieties. However, the WC of the light-coloured soybean varieties JP-2 and JP-16 did not exhibit an obvious change. For example, the WC of the yellow variety JP-16 only decreased from 18.04% to 17.41%, whereas that of the black variety JP-6 decreased from 15.97% to 9.71%. This information demonstrated that soybean seeds with dark-coloured seed coats lost water more easily than those with light-coloured seed coats. 3.6. Correlation and cluster analyses To further determine the relationships among seed viability, water content and metabolite concentrations, in-depth correla-
tion and cluster analyses were conducted based on the parameters of the different coloured soybean seeds that were stored for three years. As shown in Fig. 6A, all germination parameters (GR, GP and GI) were significantly and negatively correlated with the seed water content, isoflavones and fatty acid constituents. The water content in the seed was significantly positively correlated with the FFA constituents, especially saturated fatty acids (LA and PA). Pearson’s correlation analyses and hierarchical clustering analysis revealed the detailed relationships between the concentrations of the four groups of isoflavones and the five FFAs in the soybean seed. The results indicated strong correlations between the metabolites that participate in closely related pathways, which is consistent with a previous study (Kim et al., 2014).
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Fig. 4. Relative expression of genes involved in the isoflavone and fatty acid pathways of black and yellow soybean seed. Transcription of isoflavonoid and fatty acid genes was quantified using FPKM values. Expression levels of key genes involved in isoflavone metabolism in (a) the yellow soybean variety JP-16 and (b) the black soybean variety JP-6, and key genes involved in fatty acid metabolism in (c) the yellow soybean variety JP-16 and (d) the black soybean variety JP-6. ACP: acyl carrier protein synthase; FAS: fatty acid-CoA synthase; KCS: b-ketoacyl-CoA synthase; ACC: acetyl-CoA carboxylase; Lox: lipoxygenase; FAD: fatty acid desaturase; LPS: lipase. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. NMR colour-coded images of various coloured soybean seeds stored over different time periods. The ‘‘bright part” (high proton density) is coloured red, and the ‘‘dark part” is coloured blue. The colour scale shown beside the images indicates the relative signal intensity of the water protons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The cluster analysis results for the different coloured soybean seeds, based on the above-mentioned parameters, are shown in Fig. 6B. This heat map provides intuitive visualization of the sample information. The five soybean varieties stored for three years can be divided into three groups, i.e., high storability (JP-6), medium storability (JP-7, JP-8, JP-2), and low storability (JP-16). It can be observed that the darker the soybean seed coat colour, the
higher the storability, which is consistent with the results presented above. In addition, the water contents of stored seeds decreased with the darkening of the coat colour; this comparison is presented in Fig. 6B. Most of the tested samples contained high concentrations of FFAs, except the black soybean variety JP-6. The isoflavone concentrations in JP-6 and JP-8 were relatively low compared with the other varieties.
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Fig. 6. Multivariate statistical analysis of the metabolic characteristics associated with the storability of different soybean varieties. Heat map of correlation analysis between germination, water content, and isoflavone and fatty acid concentrations in various soybean seeds (a). Heat map of clustering analysis of metabolic characteristics of various soybean seeds (b). The colour scale shown beside the images indicates the relative signal intensity of the correlations. GR: germination rate; GP: germination potential; GI: germination index; WC: water content; OA: palmitic acid; SA: stearic acid; ALA: a-linolenic acid; OA: oleic acid; LA: linoleic acid; E: aglycones; G: b-glucosides; M: malonyglucosides; A: acetylglucosides.
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4. Discussion 4.1. Dark black soybean seeds were less inactivated Although seed aging is an irreversible physiological phenomenon, many studies have aimed to retard this deteriorative process. However, even if seeds are stored under extremely clean, dry and low temperature conditions, it is difficult to completely prevent their inactivation (Murthy et al., 2003). As a typical legume plant with a high oil content, soybean seed deteriorates easily, leading to necessary seed production and multiplication every 1– 2 years in agricultural practice. Generally, soybean germplasms with dark-coloured seed coats are more similar to the natural wild type, especially black soybean seeds, which present a ‘‘hard seed” characteristic. On the one hand, the hard seed characteristic is a type of seed dormancy; the dormancy of these hard seeds must be broken before sowing (Sun et al., 2014). Consequently, a hard seed is an undesirable characteristic for most growers. On the other hand, as an adaptive characteristic for adverse environments, a hard seed protects soybean seed against deterioration, maintaining high seed vigour over lengthy storage durations (Zhou et al., 2010). The current research indicated that although the storage treatment caused irreversible inactivation of the soybean seeds, the light-coloured seeds deteriorated more easily, whereas the dark-coloured soybean seeds maintained higher seed viability throughout the long-term storage. Dark black soybean seeds were less inactivated during storage; a possible reason may be the hardness of the seed. This characteristic includes both the physical structure and the chemical constituents, i.e., multiple hard layers and phenolics in the dark-coloured seed coat, which effectively prevent external injuries, such as oxidization and insect damage. 4.2. Storage had a strong influence on the metabolism variation As the main functional constituents of soybean seed, isoflavones and fatty acids not only represent various bioactivities in the medicinal field but also play key roles in plant physiological resistance. This study indicated that glucoside and aglycon increased with storage duration, whereas acetylglucoside and malonylglucoside decreased as a result of storage. This is consistent with previous studies (Kim, Kim, Hahn, & Chung, 2005; Lee et al., 2003), but the mechanisms involved in soy isoflavone metabolism are not well understood. Further comparative transcriptome analysis between two typical soybean seeds with different storability was conducted. The results demonstrated that isoflavone metabolism in the soybean seed did not stop during storage. The isoflavone metabolism in the light-coloured soybean seed was more susceptible to the environment, whereas the dark-coloured soybean seed appeared to be relatively stable. The expression levels of key upstream genes (PAL, CHS, CHI, and IFS) involved in isoflavone metabolism were significantly higher than those of downstream genes (UGT, AT, and MT), resulting in the transformation, and conversion, of different types of isoflavones in soybean seed being affected by storage. Furthermore, the high instability of malonyl derivatives of isoflavonoids also caused their conversion to corresponding glycosyl conjugates. Malonyl-conjugates followed by glycosyl-conjugates are the main forms of isoflavones in soybean seed, which account for more than 80% of the total content (Liu et al., 2016b). The conversion between glucoside and malonylglucoside represents the main transformation involved in isoflavone metabolism in soybean seed. The mechanism through which plant vacuoles confer stability to malonyl isoflavone conjugates remains unknown (Dastmalchi & Dhaubhadel, 2014). However, the following occurs in the conversion of malonylglucoside to glucoside: dehydration of the soybean seed during storage results in vacuolar atrophy, leading to isofla-
vone re-compartmentalization, which induces isoflavone transport from the central vacuole of the cell to the apoplast. Aglycon and glucoside are more involved in the defense interaction with the environment, compared with their corresponding malonyl or acetyl derivatives (Jones & Vogt, 2001). For example, the antioxidant activities of aglycons have been reported to be higher than those of corresponding glucoside derivatives (Mora, Payá, Ríos, & Alcaraz, 1990). This study indicated that the degree of conversion from malonylglucoside to glucoside in dark-coloured soybean seed was higher than in light-coloured seed during storage. This information implies that dark-coloured soybean seed may have a greater resistance potential based on isoflavone conversion. Storage also had a strong influence on the metabolism of fatty acids. Lipoxygenase plays a key role in seed aging (Devaiah et al., 2007). High amounts of FFAs imply the formation of peroxides and hydrolytic changes in fat components (Narayan, 1988). Compared with the up-regulation of LOX in yellow soybean seed, LOX in black soybean seed was down-regulated during the three years of storage. Significantly higher increases in FFAs were also detected in light-coloured soybean seed compared with the dark-coloured samples. The higher expression levels of late fatty acid pathway genes, especially LOX in light-coloured soybean seed, may account for the higher levels of FFAs as a result of storage. This also implies that dark-coloured soybean seed may have a greater resistance potential based on the low levels of FFAs, which indicate damage caused by peroxidation and hydrolysis (Devaiah et al., 2007). 4.3. Water content accelerates seed inactivation Several scientific hypotheses related to seed deterioration have been proposed in previous studies. A well-known theory is that free radical assault on mitochondrial membranes can be involved. This theory suggests that active oxygen species (AOS), which are free radicals generated during the oxidizing reaction in the interior of the seed, attack intracellular lipids and their constituent proteins, damaging the integrity of the plasma membrane and resulting in seed deterioration (Sanz, Pamplona, & Barja, 2006). However, cells are endowed with detoxifying enzymes and antioxidant compounds that scavenge AOS and participate in seed survival (Bailly, 2004). McDonald (1999) demonstrated that long-term storage correlates with higher accumulated AOS levels, and membrane lipid autoxidation is the main source of the accumulated AOS. Dry seed tissue metabolism can shut down due to a reduction in the mobility of molecules, which was controlled by a glass matrix structure (Bailly, 2004). Mira, González-Benito, Hill, and Walters (2010) studied the production rate of volatile peroxidation by-products of lettuce seeds during storage. These authors demonstrated that the deterioration mechanisms involved in viability loss differed depending on the seed water content; a low water content can retard seed deterioration. In this study, TD-NMR analysis indicated that the dark-coloured seed lost water more easily than the lightcoloured seed during long-term storage. This implies that darkcoloured seed has a greater resistance potential based on the high rate of water loss, which decreases AOS to mitigate oxidation. The multiple hard layers of the seed coat of dark-coloured soybean, especially those of black soybean, lead to resistance to water absorption, thus causing seed dormancy (Mullin & Xu, 2001). However, our study indicated that the hard seed of black soybean facilitated water loss and resulted in higher germination parameters. This seemingly contradictory finding suggests a two-way regulatory mechanism acts on the surface of soybean seed. Research has indicated that there are two layers of palisade cells in the soybean seed coat, one of which is located in the outer epidermal layer, referred to as the ‘‘reverse palisade layer” (Sun et al., 2014). This ‘‘reverse palisade layer” acts as a valve structure to control water circulation. For example, this layer only allows water
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dispersion, preventing the entry of water from the outside environment. This type of regulating function is especially evident in black soybean varieties. 5. Conclusions In conclusion, comparative analysis of seed vitality, metabolism and water content between dark- and light-coloured soybean seeds subjected to different storage durations revealed the partial mechanisms of seed deterioration, including the conversion of different types of isoflavones that function as infiltration regulators (Yumei & Zhou, 2011); the release of FFAs, which function as markers of lipid membrane oxidation; and the water-holding capacity of the seed vacuole, which functions as an oxidation accelerator. A combination of these factors resulted in greater inactivation of light-coloured soybean seeds, whereas dark-coloured varieties had better storability. Comparative transcriptomic and NMR relaxometry analyses revealed that the above-mentioned mechanisms may be mediated by lipoxygenase (IFS) and 2-hydroxyisoflavanone synthase (LOX) genes, and the activities of these enzymes may be regulated by the cotyledon water content. Additionally, there was a certain regularity in the storability and metabolic physiology of the different coloured soybean seeds, especially the pure coloured seed (e.g., JP-6, JP-8, JP-2, JP-16). The in-depth correlation and cluster analyses confirmed this finding. However, inconsistent results were observed for the bicoloured soybean seed (i.e., JP-7) that was checkered with black and white, which was similar to the taupe seed. The physiological and metabolic parameters of the bicoloured soybean seed (JP-7) had values that fell between the values of black (JP-6) soybean seed and brown (JP-8) soybean seed. For example, the seed vigour of JP-7 was higher than that of JP-8 but lower than that of JP-6. These results suggest that seed coat pigment is closely related to seed storability. However, determination of a quantitative relationship between pigment content and seed vitality requires further study. Consequently, further investigation of the effects of soybean seed colour and quantitative analysis of seed coat pigments should be conducted. Acknowledgements This study was financially supported by the National Natural Science Foundation of China (Grant No. 31401329) and the China Postdoctoral Science Foundation (Grant No. 2014M560724). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2016. 12.030. References Bailly, C. (2004). Active oxygen species and antioxidants in seed biology. Seed Science Research, 14, 93–107. Dastmalchi, M., & Dhaubhadel, S. (2014). Soybean seed isoflavonoids: Biosynthesis and regulation. In R. Jetter (Ed.). Phytochemicals – biosynthesis, function and application (Vol. 44, pp. 1–21). Heidelberg: Springer. De Alencar, E. R., Faroni, L. R. D. A., de Lacerda Filho, A. F., Ferreira, L. G., & Meneghitti, M. R. (2006). Influence of different storage conditions on soybean grain quality. In: Proceedings of the 9th international working conference on stored-product protection (pp. 30–37). Devaiah, S. P., Pan, X., Hong, Y., Roth, M., Welti, R., & Wang, X. (2007). Enhancing seed quality and viability by suppressing phospholipase D in Arabidopsis. Plant Journal, 50, 950–957. Gong, W., Qi, P., Du, J., Sun, X., Wu, X., Song, C., & Yang, W. (2014). Transcriptome analysis of shade-induced inhibition on leaf size in relay intercropped soybean. PLoS ONE, 9, e98465. Jones, P., & Vogt, T. (2001). Glycosyltransferases in secondary plant metabolism: tranquilizers and stimulant controllers. Planta, 213, 164–174.
113
Kim, S. H., Jung, W. S., Ahn, J. K., & Chung, I. M. (2005). Analysis of isoflavone concentration and composition in soybean [Glycine max (L.)] seeds between the cropping year and storage for 3 years. European Food Research and Technology, 220, 207–214. Kim, J. J., Kim, S. H., Hahn, S. J., & Chung, I. M. (2005). Changing soybean isoflavone composition and concentrations under two different storage conditions over three years. Food Research International, 38, 435–444. Kim, J. K., Kim, E. H., Park, I., Yu, B. R., Lim, J. D., Lee, Y. S., ... Chung, I. M. (2014). Isoflavones profiling of soybean [Glycine max (L.) Merrill] germplasms and their correlations with metabolic pathways. Food Chemistry, 153, 258–264. Kumar Dixit, A., Kumar, V., Rani, A., Manjaya, J. G., & Bhatnagar, D. (2011). Effect of gamma irradiation on lipoxygenases, trypsin inhibitor, raffinose family oligosaccharides and nutritional factors of different seed coat colored soybean (Glycine max L.). Radiation Physics and Chemistry, 80, 597–603. Lee, S. J., Ahn, J. K., Kim, S. H., Kim, J. T., Han, S. J., Jung, M. Y., & Chung, I. M. (2003). Variation in isoflavone of soybean cultivars with location and storage duration. Journal of Agricultural and Food Chemistry, 51, 3382–3389. Li, T., Rui, X., Tu, C., Li, W., Wang, K., Huang, L., & Dong, M. (2015). NMR relaxometry and imaging to study water dynamics during soaking and blanching of soybean. International Journal of Food Engineering, 12, 181–188. Ling, L., Jiafeng, J., Jiangang, L., Minchong, S., Xin, H., Hanliang, S., & Yuanhua, D. (2014). Effects of cold plasma treatment on seed germination and seedling growth of soybean. Scientific Reports, 4, 5859. Liu, J., Gui, J., Gao, W., Ma, J., & Wang, Q. (2016a). Review of the physiological and biochemical reactions and molecular mechanisms of seed aging. Acta Ecologica Sinica, 36. Liu, J., Yang, C.-Q., Zhang, Q., Lou, Y., Wu, H.-J., Deng, J.-C., ... Yang, W.-Y. (2016b). Partial improvements in the flavor quality of soybean seeds using intercropping systems with appropriate shading. Food Chemistry, 207, 107–114. Ma, L., Li, B., Han, F., Yan, S., Wang, L., & Sun, J. (2015). Evaluation of the chemical quality traits of soybean seeds, as related to sensory attributes of soymilk. Food Chemistry, 173, 694–701. McDonald, M. B. (1999). Seed deterioration: Physiology, repair and assessment. Seed Science and Technology, 27, 177–237. Midorikawa, K., Kuroda, M., Terauchi, K., Hoshi, M., Ikenaga, S., Ishimaru, Y., ... Asakura, T. (2014). Additional nitrogen fertilization at heading time of rice down-regulates cellulose synthesis in seed endosperm. PLoS ONE, 9, e98738. Mira, S., González-Benito, M. E., Hill, L. M., & Walters, C. (2010). Characterization of volatile production during storage of lettuce (Lactuca sativa) seed. Journal of Experimental Botany, 61, 3915–3924. Mora, A., Payá, M., Ríos, J. L., & Alcaraz, M. J. (1990). Structure-activity relationships of polymethoxyflavones and other flavonoids as inhibitors of non-enzymic lipid peroxidation. Biochemical Pharmacology, 40, 793–797. Mullin, W. J., & Xu, W. (2001). Study of soybean seed coat components and their relationship to water absorption. Journal of Agricultural and Food Chemistry, 49, 5331–5335. Murthy, U. M. N., Kumar, P. P., & Sun, W. Q. (2003). Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: Lipid peroxidation, sugar hydrolysis, Maillard reactions and their relationship to glass state transition. Journal of Experimental Botany, 54, 1057–1067. Narayan, R. (1988). Changes in the quality of soybean during storage. Part 1—Effect of storage on some physico-chemical properties of soybean. Food Chemistry, 27, 13–23. Narayan, R., Chauhan, G., & Verma, N. (1988). Changes in the quality of soybean during storage. Part 2—Effect of soybean storage on the sensory qualities of the products made there from. Food Chemistry, 30, 181–190. Sanz, A., Pamplona, R., & Barja, G. (2006). Is the mitochondrial free radical theory of aging intact? Antioxidants & Redox Signaling, 8, 582–599. Sun, X., Wang, Z., Li, S., Men, F., Wang, S., & Zhang, J. (2014). Progress on formation mechanism and breaking methods of hard seed in soybean. Soybean Science & Technology, 3, 23–27. Wang, T., Harp, T., Hammond, E. G., Burrisa, J. S., & Fehr, W. R. (2001). Seed physiological performance of soybeans with altered saturated fatty acid contents. Seed Science Research, 11, 93–97. Wang, F., Wang, R., Jing, W., & Zhang, W. (2012). Quantitative dissection of lipid degradation in rice seeds during accelerated aging. Plant Growth Regulation, 66, 49–58. Wu, K., Xiao, S., Chen, Q., Wang, Q., Zhang, Y., Li, K., ... Chen, L. (2013). Changes in the activity and transcription of antioxidant enzymes in response to al stress in black soybeans. Plant Molecular Biology Reporter, 31, 141–150. Xia, J., Sinelnikov, I. V., Han, B., & Wishart, D. S. (2015). MetaboAnalyst 3.0—making metabolomics more meaningful. Nucleic Acids Research, 43, W251–W257. Xiao, Z., Mak, A., Koch, K., & Moore, R. B. (2013). A molecular complex of bovine milk protein and oleic acid selectively kills cancer cells in vitro and inhibits tumour growth in an orthotopic rat bladder tumour model. BJU International, 112, E201–E210. Yumei, W. U., & Zhou, Q. (2011). Effects of seed soaking with soybean isoflavones on soybean seedlings under salt stress. Acta Ecologica Sinica, 31, 6669–6676. Zhang, M. (1994). A mechanism of seed deterioration in relation to the volatile compounds evolved by dry seeds themselves. Seed Science Research, 4, 49–56. Zhang, T., Kawabata, K., Kitano, R., & Ashida, H. (2013). Preventive effects of black soybean seed coat polyphenols against DNA damage in Salmonella typhimurium. Food Science and Technology Research, 19, 685–690. Zhou, S., Sekizaki, H., Yang, Z. H., Sawa, S., & Pan, J. (2010). Phenolics in the seed coat of wild soybean (Glycine soja) and their significance for seed hardness and seed germination. Journal of Agricultural and Food Chemistry, 58, 10972–10978.