Abstract

   γ-aminobutyric acid (GABA) has been reported to improve stress
   resistance in plants. Nonetheless, little is known about the effects of
   GABA on the nutritional quality and regulatory mechanisms of edamame.
   Therefore, we analyzed the flavonoid and amino acid (AA) metabolism and
   the effects of GABA on the nutrient content of edamame seeds through
   physiological and metabolomic analyses. Exogenous GABA increased
   endogenous GABA metabolism and GABA transaminase activity and enhanced
   the oxoglutarate content, which entered into nitrogen metabolism and
   increased the activity and expression of nitrogen metabolism-related
   enzymes, to accumulate AAs and bioactive peptides. Meanwhile, exogenous
   GABA induced the metabolism of flavonoids, including total flavonoids,
   anthocyanins, 6′'-o-malonyglycitin, glycitin, ononin, cyanin, and
   ginkgetin, by increasing the activity and expression of flavonoid
   biosynthetic enzymes. This is the first study to reveal that GABA
   effectively improves the nutritional quality of edamame through the
   accumulation of AAs, bioactive peptides, isoflavones, anthocyanins,
   sugars, and organic acids.

Introduction

   Edamame [Glycine max (L.) Merr.], also called “vegetable soybean,”
   “green edamame,” or “vegetable-type soybean,” is harvested when the
   pods and seeds are still green (R6 growth stage) ([47]Fehr, 1971).
   Edamame is popularly consumed as a cooked or roasted snack and is also
   consumed as an addition to salads, soups, stews, stir-fried dishes,
   processed sweets, and desserts ([48]Song et al., 2013).

   With the improvement in living standards, nutrition has garnered
   increased scientific attention. Previous studies revealed that the
   content of nutrients, including free amino acids
   (4.581–10.180 mg g^−1), sucrose (8.2 g 100 g^−1), isoflavones (27.02 mg
   100 g^−1), and oil (212.6 g kg^−1), in edamame seeds was higher than
   that in soybean grains ([49]Song et al., 2013, [50]Carneiro et al.,
   2021, [51]Jiang et al., 2020).

   Amino acids are the primary metabolites that play a vital role in
   growth, development, and health maintenance and serve as building
   blocks of proteins and polypeptides exhibiting a wide range of
   biological activities, including hypolipidemic, anti-diabetic,
   anti-hypertensive, and anti-cancerous properties ([52]Daliri, Oh, &
   Lee, 2017). Additionally, exogenous reagents have been known to promote
   the accumulation of amino acids by affecting the expression and
   activity of enzymes involved in nitrogen metabolism ([53]Wang et al.,
   2014, [54]Wang et al., 2022, [55]Zhang et al., 2020).

   Flavonoids, including isoflavonoids, chalcones, flavones, flavonols,
   flavanones, and anthocyanins, are important phenolic secondary
   metabolites in plants synthesized through the phenylpropane metabolic
   pathway ([56]Chen, Zhang, Zhang, Li, & Ma, 2019). They promote disease
   resistance in plants and exhibit anti-oxidant, anti-cancerous, and
   anti-aging properties in humans ([57]Chen et al., 2019). However, they
   can also be modulated by multiple exogenous agents, as they are
   synthesized using a complex biochemical process involving different
   signaling pathways ([58]Zhao et al., 2021, [59]Jia et al., 2019).

   [60]Song et al. (2013) determined that edamame contained 23 amino
   acids, including all the essential amino acids. In addition, edamame is
   an excellent dietary source of natural flavonoids ([61]Wang et al.,
   2018), whose content was higher in edamame than in other legumes
   ([62]Kim et al., 2006). Edamame is also rich in organic acids, vitamins
   (C, E, and B1), minerals, phytochemicals, and other active compounds
   that have the potential to reduce the risk of several diseases such as
   cardiovascular disease, cancer, and osteoporosis ([63]Jiang et al.,
   2020). Nevertheless, the mechanisms underlying the regulation of
   nutritional content in edamame have received limited attention.
   Therefore, we focused on the nutritional quality and regulatory
   mechanisms in edamame.

   γ-aminobutyric acid (GABA) is a ubiquitous non-proteinogenic amino acid
   found in bacteria, fungi, plants, and animals and exhibits
   anti-hypertensive and anti-stress effects on human health ([64]Ma,
   Wang, Chen, Gu, & Yang, 2018). As a functional food ingredient, GABA
   has been enriched in a myriad of foods, such as cereal-based foods,
   dairy products, beverages, meat, vegetables, and legumes, through
   lactic acid bacteria fermentation or manipulation of plant food
   material ([65]Diana, Quílez, & Rafecas, 2014). In addition, GABA has
   been used as a food additive or supplement ([66]Boonstra et al., 2015).
   While GABA constitutes a considerable portion of the free amino acid
   pool in plants, it is mainly synthesized and metabolized via the GABA
   shunt and catalyzed by glutamate (Glu) decarboxylase (GAD) and GABA
   transaminase (GABA-T) ([67]Ma et al., 2018).

   Exogenous GABA enhances resistance to hypoxia in melon and storage
   performance of postharvest citrus fruits by promoting the tricarboxylic
   acid (TCA) cycle and the accumulation of endogenous amino acids
   ([68]Wang et al., 2014, [69]Sheng et al., 2017). Moreover, as a key
   signaling molecule, GABA is involved in NaCl stress in barley ([70]Ma
   et al., 2018) and soybean ([71]Zhao et al., 2021), suggesting that
   exogenous GABA can upregulate the activity and expression of
   phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H),
   and 4-coumarate coenzyme A ligase (4CL) to accumulate anthocyanins,
   phenolic compounds, and flavonoids. In addition, exogenous GABA can
   enhance the content of amino acids and soluble sugars in snap beans to
   ameliorate drought stress ([72]Abd El-Gawad et al., 2021). However,
   there have been no reports on the direct effects of GABA on the quality
   of crops, especially edamame.

   Therefore, we determined the effects of GABA on flavonoid and amino
   acid metabolism and the contents of sugars, vitamin C, organic acid,
   and other substances in edamame seeds using physiological and
   metabolomic methods for the first time to explore the how GABA
   regulates the nutritional quality of edamame.

2. Materials and methods

2.1. Plant growth conditions

   Six treatments were established to screen for suitable concentrations
   of GABA (CAS NO.56–12-2, Sigma-Aldrich Co., ltd. CA). The leaves of
   edamame at the R1 stage were sprayed with 0, 2.5, 5, 10, 20, and
   30 mmol/L (mM) of GABA. For each treatment, the leaves of the second or
   the third leaves from the top were harvested with three replicates
   after 6 days. Among them, the content of nutrients (including GABA,
   sucrose, and vitamin C), and the enzyme activity of glutamine
   synthetase (GS), glutamic-oxaloacetic transaminase (GOT), glutamic acid
   decarboxylase (GAD), and γ-aminobutyric acid transferase (GABA-T)
   related to nitrogen metabolism in edamame plants were determined (data
   not shown), and foliar application of 10 mM GABA exerted maximum
   benefits for phenotype. Subsequently, 10 mM GABA was used for
   experiments at the R5 stage to determine the effects of GABA on the
   nutrient content of edamame. The enzyme activity of leaves and
   substance content of seeds of plants treated after 6, 9, 12, 18, and
   24 days were assayed. In addition, at samples at 12 h, 24 h and 3 d
   after treatments were used for quantitative reverse transcription
   polymerase chain reaction (qRT-PCR) analysis, and stored at −80 °C
   until analysis. Three biological replicates were used for all the test.

2.2. Determination of sugar content

   All substances were extracted from fresh seeds (2.0 g). The content of
   sucrose, glucose, and fructose was assayed based on the methodologies
   of [73]Du et al., 2020, [74]Li et al., 2016. Sample was extracted with
   80 % (v/v) ethanol (CAS NO. 64–17-5, Sigma-Aldrich Co., ltd. CA) at
   80 °C for 30 min, followed by centrifugation at 10,000 × g for 10 min.
   The residue was extracted three times with 80 % ethanol. The three
   supernatants were combined and added with 80 % ethanol to a total
   volume of 5 mL, and then the content of sucrose, glucose, and fructose
   were determined using spectrophotometric method at the wavelength of
   A[480], A[625], and A[291] nm, respectively.

2.3. Assay of the content of Vc and glutathione (GSH)

   The content of Vc and GSH in edamame seeds were estimated by
   homogenizing fresh sample tissue at 5 % (w/v) metaphosphoric acid (CAS
   NO. 37267–86-0, Sigma-Aldrich Co., ltd. CA) and 5 % (w/v)
   sulpho-salicylic acid (CAS NO. 97–05-2, Sigma-Aldrich Co., ltd. CA),
   respectively (The content of GSH and Vc was estimated following the
   method of [75]Jiao, Yang, Zhou, and Gu (2016).

2.4. Detection of the content of amino acids and related enzyme activity

   The concentration of GABA and free amino acids was estimated following
   the method of [76]Priya et al. (2019). The content of free amino acids
   was determined with ninhydrin (CAS NO. 485–47-2, Sigma-Aldrich Co.,
   ltd. CA) reagent according to the method of [77]Wang et al., 2014),
   respectively.

   All samples for determination of enzymes activity were extracted from
   fresh leaves (0.3 g). The activity of nitrate reductase (NR), glutamine
   synthetase (GS), glutamate synthase (GOGAT), and glutamate
   dehydrogenase (GDH) was determined according to the method of [78]Wang
   et al. (2014). The activity of glutamic-oxaloacetic transaminase (GOT)
   and glutamic-pyruvic transaminase (GPT) was assayed according to
   [79]Kaur’ s. (2015) method. The activity of glutamate decarboxylase
   (GAD) and GABA transaminase (GABA-T) was assayed according to [80]Li et
   al. (2016).

2.5. Determination of flavonoids and related enzyme activity

   The content of total flavonoid and total anthocyanins was determined
   using the method of [81]Jia et al. (2019). The activity of
   phenylalanine ammonia lyase (PAL), chalcone isomerase (CHI),
   anthocyanidin synthase (ANS), dihydroflavanol 4-reductase (DRF), and
   UDP-glycose flavonoid glycosyltransferase (UFGT) was assayed following
   the methods described by [82]Chen et al. (2019). The activity of
   cinnamate-4-hydroxylase (C4H) and 4-coumarateCoA ligase (4CL) was
   measured according to [83]Ma et al. (2018). The activity of isoflavone
   synthase (IFS) and chalcone synthase (CHS) was determined by
   enzyme-linked immunoassay, using the IFS and CHS assay system kit (GE
   Healthcare), as described in the manufacturer’s instructions.

2.6. Sample preparation for LC–MS

   The samples of edamame seeds treated with H[2]O (CK) and exogenous GABA
   for 18 days were grounded and mixed and then sent to BmK company for
   nontargeted LC-mass spectrometry (MS) detection. Each treatment was set
   up for 5 replicates. Data analysis was performed using BMK Cloud
   ([84]www.biocloud.net).

2.7. RNA extraction and qRT-PCR assays

   TRIzol® reagent (Invitrogen, Carlsbad, CA, United States) was used to
   extract total RNA. One microgram per RNA sample was used as the
   template for synthesis of the first-strand cDNA, using the ReverTra
   Ace™ qPCR RT Master Mix with gDNA Remover (TOYOBO Co., Osaka, Japan).
   Next, qRT-PCR was performed using SYBR® Select Master Mix qRT-PCR
   System (Takara) on an optical 96-well plate. Actin was used as an
   internal reference. All the primers used for gene expression analysis
   were shown in supplementary [85]Table S1. The relative expression level
   was calculated with the formula 2^−ΔΔCT ([86]Livak & Schmittgen, 2001).
   Three independent biological replicates were analyzed.

2.8. Statistical analysis

   At least three independent experiments were performed for each
   treatment. Data were expressed as mean ± standard error (SE) after a
   one-way analysis of variance (ANOVA) using the SPSS 17.0 program (SPSS
   Inc. Chicago, IL, United States). The data were statistically analyzed
   using the multiple range test of Duncan (P < 0.05). The PCA and OPLS-DA
   were performed using SIMCA v13.0.3 software (Umetrics, Umea, Sweden).

3. Results and discussion

3.1. Effects of exogenous GABA on nutritional content in edamame

   Edamame contains nutrients such as carbohydrates, proteins, vitamins,
   minerals, and flavonoids ([87]Jiang et al., 2020). Previous studies
   suggest that exogenous GABA can enhance the nutrient content in snap
   beans to improve drought stress resistance ([88]Abd El-Gawad et al.,
   2021, [89]Sheng et al., 2017) and might positively affect the storage
   quality of postharvest apple and citrus fruits ([90]Li et al., 2021).
   In our study, we determined the contents of nine nutrients (GABA, free
   amino acids, glutathione (GSH), flavonoids, anthocyanins, vitamin C
   (Vc), glucose, sucrose, and fructose) in edamame seeds and revealed
   that exogenous GABA contributed to nutrient accumulation in edamame,
   especially endogenous GABA and glucose ([91]Fig. 1).

Fig. 1.

   [92]Fig. 1
   [93]Open in a new tab

   The contents of GABA (A), free amino acids (B), GSH (C), flavonoid (D),
   anthocyanins (E), Vc (F), glucose (G), sucrose (H), and fructose (I))
   in edamame fruit after 6, 9, 12, 18, and 24 d of H[2]O (control, CK)
   and GABA treatment. Data represent mean ± SE (n = 3). Asterisks (*)
   represent significant differences (Student’s t-test, P < 0.05) between
   control and GABA treatments.

   Compared to the control, the content of endogenous GABA significantly
   increased by 22.4 %, 29.8 %, 59.6 %, and 18.2 % after 6, 9, 12 and 18 d
   of GABA foliar application, and glucose significantly increased by
   38.6 %, 25.2 %, and 19.5 % after 9, 18, and 24 d of exogenous GABA
   treatment, respectively ([94]Fig. 1A, 1G). In addition, the content of
   GSH increased by 21.7 % and 22.0 %, anthocyanins by 23.3 % and 88.0 %,
   and Vc by 22.0 % and 39.4 % after 18 and 24 d of exogenous GABA
   treatment, respectively ([95]Fig. 1C, 1F). Moreover, compared to the
   control, the content of free amino acids markedly increased by 33.4 %
   and 92.4 % after 6 and 18 d of treatment, respectively ([96]Fig. 1B).
   Exogenous GABA treatment remarkably enhanced total flavonoids by 38.0 %
   after 18 d and fructose content by 18.8 % after 12 d of treatment
   ([97]Fig. 1D, 1I).

   Overall, the contents of GABA, free amino acids, GSH, flavonoids,
   anthocyanins, Vc, and glucose significantly increased after 18 d of
   treatment, with the former five reaching maxima at 18 d after exogenous
   GABA treatment ([98]Fig. 1A–G). Therefore, LC-MS analysis was performed
   using seeds after 18 d of treatment.

3.2. Non-targeted metabolomics analysis of edamame

   To further determine the effects of exogenous GABA on the nutritional
   quality of edamame, we used LC–MS to identify the metabolites present
   in edamame seeds after 18 d of treatment with H[2]O (control, CK) and
   GABA. The total ion current (TIC) for the QC sample showed that the TIC
   metabolite detection curves exhibited a high degree of overlap
   (Supplementary [99]Figures 1A, 1B), indicating that metabolite
   extraction and detection were reliable. This was important because
   unsupervised principal component analysis (PCA) of metabolites reflects
   the variability between and within sample groups. The scores of the sum
   of the first and second principal components of CK and GABA groups were
   greater than 75 % ([100]Fig. 2A, 2B, Supplementary [101]Figures 1C,
   1D). Furthermore, OPLS-DA models indicated that R2Y was greater than
   0.99 and Q2Y was greater than 0.85 in the positive and negative ion
   modes, which indicated good fitness and prediction of the experiment,
   respectively ([102]Fig. 2C, 2D). In addition, the slope of the Q2Y
   fitting regression curve was greater than zero in permutation,
   illustrating the statistical validity of the OPLS-DA model ([103]Fig.
   2E, 2F).

Fig. 2.

   [104]Fig. 2
   [105]Open in a new tab

   Principal component analysis and PCA 2D score of CK and GABA-treated
   samples (A, B). The X-axis represents the first principal component,
   and Y-axis represents the second principal component. Point diagrams of
   OPLS-DA of the samples and model permutation verification diagram
   (C–F). A, C, and E are positive ion modes, whereas B, D, and F are
   negative ion modes. Note: Blue and red dots represent R2Y and Q2Y of
   the model after replacement, respectively. The two dotted lines
   indicate the regression curves of R2Y and Q2Y fitting in the
   permutation verification diagram. (For interpretation of the references