Effect of Allelic Variation of β-conglycinin α-subunit Locus cgy-2 on Soybean (Glycine max) Quality I. Evaluation of Free Amino Acid Content

Qiu Zhen-dong, Luo Ting-ting, Song Yan-ru, Ma Chong-xuan, Tian Yu-su, Zhou Jin-tao, Song Bo, and Liu Shan-shan

Key Laboratory of Soybean Biology in Chinese Ministry of Education (Key Laboratory of Soybean Biology and Breeding/Genetics of Chinese Agriculture Ministry), Northeast Agricultural University, Harbin 150030, China

Abstract: The effects of the deficiency of the allergenic α-subunit on soybean amino acid (AA) composition were studied using the cultivar Dongnong 47 (DN47) as a genetic background. The near-isogenic line (NIL) NIL-DN47-Δα of DN47, with an introgression of the α-null trait allele from the high protein donor parent RiB, was created by marker assisted background selection and used to investigate the AA content and nutritional quality. The contents of crude protein, the total AAs, the total essential amino acids (EAAs) and sulfur-containing (Met and Cys) AAs increased by 4.11%, 4.16%, 5.20% and 11.96%, respectively in NIL-DN47-Δα compared with DN47. Analyses of the total EAAs (TEAAs) and the EAA index (EAAI) revealed that both parameters in NIL-DN47-Δα were higher than those in DN47. The null-allele of the α-subunit positively affected the AA scores. The quantitative changes in free AAs (FAAs) in the developing seeds of NIL-DN47-Δα and DN47 were compared as of 15 days after flowering (DAF) until maturity. The results showed that the total FAA content in NIL-DN47-Δα was significantly higher than that in the DN47 throughout the late maturation stage (40-60 DAF) of seeds. The high concentration of the FAAs in cgy-2 mutant seeds was a consequence of the high rates of synthesis and/or accumulation of individual FAAs during seed maturation where 25 DAF was an important turning point in the accumulation of the FAAs. The FAA contents of single soybean α-null, double (α+α′) and triple (α+α′+group I)-null mutant combination lines were investigated. In all of these combinations, introduction of the cgy-2 gene invariably raised the FAA content of mature seeds above that of the DN47. In summary, the enhanced protein quality in cgy-2 mutants resulted from several factors. (1) There was a general increase in the contents of most AAs and FAAs in NIL-DN47-Δα. (2) The induced synthesis of free Arg contributed effectively to the high FAAs of various storage-protein-deficiency mutants. For example, in the S2 (null α, group I), the free Arg content was seven times as much as that of DN47, accounting for more than half of the total FAA content in the seed. (3) The increase of sulfur-containing AAs in the α-null type NIL mainly resulted from elevated Met content. These data suggested that the cgy-2 mutation might improve the protein quality of soybean seeds and that lacked of the allergenic α-subunit resulted in increased the FAA content.

Key words: soybean (Glycine) β-conglycinin, α-subunit null mutant, free amino acid, arg-overproducing, seed storage protein

Introduction

Soybean plants produce the highest protein yield per unit area of land and have the highest protein content among all the seed crops. Soybean is widely used in manufacturing many processed food, serving as a raw material for numerous products (Young, 1991; Friedman and Brandon, 2001). Accordingly, it is essential to develop new or modified seed components with enhanced proteins and oil.

The protein contents of soybean seeds and the subunit composition of storage proteins influence both processing of seed derived products and their final nutritional quality. Glycinin (11S globulin) andβ-conglycinin (7S) are the two major proteins accounting for up to 70% of total soybean seed proteins. Glycinin is a hexamer with a molecular mass of about 350 ku.It comprises five major subunits classified into two groups based on sequence similarity and size, namely group I (A1aB1b, A1aB2and A2B1a) and group II (A3B4and A5A4B3) (Moriet al., 1981; Nielsen, 1985; Nielsenet al., 1989; Yagasaki, 1996). Theβ-conglycinin is a trimeric glycoprotein composed of the three subunitsα′,αandβwithpIvalues of 4.9, 5.2 and 5.7, respectively (Thanh and Shibasaki, 1996). Glycinin is relatively richer in sulfur-containing amino acids as compared toβ-conglycinin (Koshiyama, 1968). Moreover, glycinin shows superior functional gel and film properties to those of 7S globulin (Saio and Watanabe, 1978). Theα,α′ andβ-subunits ofβ-conglycinin are known food allergens (Ogawaet al., 1995; Krishnanet al., 2009; Holzhauseret al., 2009). Genetic studies (Kitamuraet al., 1984; Tsukadaet al.,1986; Takahashiet al., 1996) have demonstrated that theα′ and α-subunits are controlled by respective single alleles. The presence or absence of theα′-subunit is independent from that of the α-subunit. The expression of theα′ or α-subunit is dominant to the lack of each. The gene symbolsCgy1/cgy1 andCgy2/cgy2 were assigned to the presence/absence of theα′ andα-subunits, respectively (Kitamuraet al., 1984; Tsukadaet al., 1986). However, limited evidence has been reported as to single gene effectiveness of eachβ-conglycinin subunit and the molecular mechanism of regulation of the gene of each allergen subunit ofβ-conglycinin remains unclear. It is suggested that increasing the content of 11S at the expense of 7S globulin may enhance the nutritional value of soybean. Therefore, optimizing the 11S:7S ratio may improve soybean protein utilization in manufacturing various kinds of food (Ogawaet al., 1989; Ogawaet al., 2000). By manipulating the variant alleles identified, it has been possible to breed soybean varieties with markedly modified protein composition, ranging from extremely high to extremely low 11S:7S ratios. This leads to improved nutritional values and foodprocessing properties (Takahashiet al., 2004). Mutant genes of various storage protein subunits have been used to modify seed protein composition and produce allergen subunit-null mutants that eliminate the undesirable allergens (Ogawaet al., 2000; Songet al., 2014). Over the past three decades, efforts to develop 7S-low type soybean lines had led to the availability of various 7S or 11S globulin protein subunit null varieties in the soybean germplasm (Yagasakiet al., 1996; Songet al., 2014; Kitamura and Kaizuma, 1981; Haradeet al., 1983; Odanaka and Kaizuma, 1989; Takahashiet al., 1994; Hajikaet al., 1996). Despite the absence of the two major storage protein subunits, most mutant varieties grew and reproduced normally and the nitrogen contents of the dry seeds were similar to (or even higher than) those of the wild-type cultivars (Takahashiet al., 2003). Theβ-conglycinin-deficient mutants have been proven to be unique in their high nutritional value and low allergenic risk (Takahashiet al., 2004; Ogawaet al., 1989; Ogawaet al., 2000; Hajikaet al., 1998; Hajikaet al., 2009).

Soybean amino acids not only have a high nutritive value, but also provide several health benefits, such as reduction in blood cholesterol (Gibbset al., 2004; Potter, 1995), decreasing the risk of mutagenicity (Leeet al., 2005), reduction of coronary heart disease (Andersonet al., 1995) and possible control of obesity (Allisonet al., 2003; Fontaineet al., 2003). Some amino acids, such as Tyr, Met, His, Lys and Trp, may act as antioxidants (Saitoet al., 2003). Kimet al. (1999) reported that soybean glycopeptides comprising Asp, Glu, Pro, Gly and Leu have strong cytotoxic activity against cancer cells. In contrast to the reported extensive studies on various properties of the AAs of soybean seeds in the form of storage proteins, little is known about the FAA in soybean seeds. The increase in the FAA content of storage protein-deficient seeds has been described (Songet al., 2014; Takahashiet al., 2003; Kitaet al., 2010).

Compensative accumulation of the FAAs is first observed in a null mutation lacking both glycinin andβ-conglycinin (Takahashiet al., 2003). The intrinsic null mutation responsible for storage protein deficiency affectes the synthesis of inherent alternative nitrogen reservoirs, such as free Arg (Kitaet al., 2010). Similar results are observed in a related study where the seeds of the mutant soybean variety RiB, which lacksα′ andαsubunits ofβ-conglycinin as well as most glycinin acidic subunits, maintained nitrogen reserves with higher FAA contents and shows a particularly enriched Arg pools (Songet al., 2014). This suggests that reduction in storage protein quantity is compensated for by activation of alternative nitrogen assimilation pathways resulting in the accumulation of extra amounts of the FAAs.

In this report, data were presented on the contents of the AAs and the FAAs of a soybeanα-null type near isogenic line (NIL) and the developmental changes in the synthesis of the FAAs in the seeds ofα-subunitdeficient NIL lines were reported. Temporal differences in the rates of the FAA accumulation between theα-null mutant NIL-DN47-Δαand the wild type Dongnong 47 (DN47) were observed. The study was extended to include other storage protein subunitdeficient mutant combinations. In all the combinations studied, the presence ofcgy-2 gene raised the levels of the FAAs in the single S1 (nullα), double S2 (nullα, group I) and triple S6 (nullα,α′, group I) mutants above those of DN47. The present study provided insights into understanding the effects of thecgy-2 gene on the FAA accumulation as well as the AA quality in soybean seeds. These results provided scientific basis and theoretical supports for scientific and rational utilization of soybean protein with deletion of sensitizing proteinα-subunit, improvement of nutritional value and mutant soybean subunit.

Materials and Methods

Experimental design

This study was a part of the program for developing soybean allergen subunit-deficient mutant varieties, conducted at the Key Laboratory of Soybean Biology and Breeding/Genetics of Chinese Agriculture Ministry. Based on a triple-screening procedure comprisingα-null phenotype screening, marker-assisted background selection and stringent phenotypic selection; various backcross and three-way-cross hybrids containing thecgy-2 gene were obtained (Table 1). The storage protein subunit-deficient mutant populations developed from the above mentioned researches were used to evaluate the effect of thecgy-2 gene on the FAA contents of soybean seeds in the present study.

Plant materials and field experiments

The recurrent parent Dongnong 47 (DN47) containing all the relevant protein subunits (Fig. 1, Lanes 1 and 2) was used as a control. The RiB genotype which lackedα′ andα-subunits ofβ-conglycinin and glycinin group I(Fig. 1, Lanes 5 and 6) was used as thecgy-2 gene donor parent. Six storage protein subunit-deficient combination mutants containing thecgy-2 gene(Fig. 2) (generations were listed in Table 1) were used to analyze the effects of thecyg-2 gene in different genetic back-grounds.

The near isogenic line NIL-DN47-Δα(Fig. 1, Lanes 3 and 4) carrying thecyg-2 allele (conferringα-null) was used to study the accumulation pattern of FAAs during seed development in the storage proteindeficientα-null type soybean mutants. The NILs were developed through marker-assisted backcrossing of thecgy-2 from RiB into the DN47 background for four times to produce a BC4 population. The NIL line cb77-11-14 (BC4F2) showed 99.49% recurrent parent genome recovery (RPGR) content (based on the whole-genome marker analysis) (Songet al., 2014). The recessive homozygote (cyg-2/cyg-2) seeds from this line were screened by the SDS-PAGE and were self-crossed twice to obtain the cb77-13-12088-2-8 (BC4F4) line (hereafter referred to as NIL-DN47-Δα, Table 1). The line NIL-DN47-Δαdiffered from the DN47 control only in the introgressed donor DNA fragment containing thecgy-2 gene (Fig. 3). Therefore, any observable phenotypic differences were expected to be results of thecgy-2 gene.

All the soybean (Glycine maxL. Merrill) varieties and lines used in this study were sown in a field, at the agricultural experiment station of the Northeast Agriculture University, and harvested at maturity in 2014. About 45 days after sowing, the fully opened flowers were marked individually with tags at the fourth, fifth, sixth or seventh nodes on NIL-DN47-Δαand DN47. The tagged pods developing from the flowers of these nodes were periodically harvested at different stages of seed development, namely at 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 days after flowering (DAF) (Fig. 4A). The full seeds (cotyledons and seed coats) of a given stage were pooled and stored at -80℃ for future use. Seeds of odd sizes were excluded from sampling.

Table 1 Agronomic characteristics, seed protein and oil content and 11/7S ratios in recurrent parent DN47 and mutant lines used in this study

Analysis of seed protein subunit composition

The seed proteins from DN47 and NIL-DN47-Δαwere extracted at various stages of maturation (Fig. 4B)and analyzed by the SDS-PAGE as described by Laemmli (1970). The total seed proteins were extracted from small portions of the cotyledon tissues in the SDS sample buffer (2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 5 mmol ? L-1urea, 62.5 mmol ? L-1Tris amino methane) and then centrifuged for 10 min at 12 000×g. Samples of 10 μL volume of supernatants were separated on 4.5% stacking and 12.5% separating polyacrylamide gels and stained with Coomassie Brilliant Blue R 250.

The 11S/7S ratios were determined by densitometric analyses following electrophoresis. Table 1 summarized the differences in protein subunit composition and the ratios of glycinin toβ-conglycinin in the seven soybean materials.

Fig. 1 SDS-PAGE gel of recurrent parent Dongnong 47 (DN47), α-null type near isogenic line NIL-DN47-Δα, and α-null trait donor parent (RiB) showing protein subunit compositionM, Marker. Positions of α, α', β of β-conglycinin, and glyciningroup I subunits are shown. Glycinin-group I includes A1aB2, A1bB1b and A2B1a.

Analysis of amino acids

The total nitrogen contents of the seeds were measured using the Micro-Kjeldahl method (Foss, 2300 Kjeltec Analyzer Unit). The crude protein content was determined by calculating the nitrogen content and then multiplying the result by the conventional factor 6.25. The total AA contents were obtained from hydrolysis of the seed meals in 6 mmol ? L-1HCl for 22 h in sealed evacuated tubes at a constant boiling temperature of 110℃. An amino acid analyzer (Hitachi L-8800; Tokyo, Japan) was used to determine the AA composition of the hydrolysates.

The free AAs were extracted from 5 g samples of the seed meals. Seed meals (seeds were sampled using a four-fold classification, fully dried with mill grinding through a 0.25 mm sieve and thoroughly mixed) were finely homogenized in 30 mL of sulfosalicylic acid (100 mL for each 10 g seed) and disrupted ultrasonically for 30 min. The supernatants were centrifuged at 5 000×g for 5 min. The resultant supernatants were filtered through a 25 mL GD/X sterile disposable syringe. A Hitachi L-8800 amino acid analyzer was used to analyze the AAs in the filtrates.

Fig. 2 SDS-PAGE gel of recurrent parent 'DN47', α-null trait donor parent 'RiB', and six null soybean genotypes showing protein subunit compositionM, Marker. DN47 (Dongnong 47); RiB (null α, α' and Group I); S1 (null α); S2 (null α, glycinin-group I); S3 (null α); S4 (null α, glycinin-group I); S5 (null α', α); S6 (null α', α, glycinin-group I). Glycinin-group I includes A1aB2, A1bB1b and A2B1a.

Fig. 3 Dongnong 47 (DN47) and near-isogenic line 'NIL-DN47-Δα' simple sequence repeat linkage map for chromosome 20 (Linkage Group I) developed from a BC4F4 ('DN47'×'RiB') population (recurrent parent genome recovery=99.51%) (Song et al., 2014)Map distances are in centimorgans (cM). Introgressed segment containing cgy-2 gene for 'NIL-DN47-Δα' is represented by black bar.

The AA concentrations of the tested protein samples were compared with a scoring pattern. The concentrations were expressed as grams of amino acid/16 gN in the test protein, divided by grams of amino acid/16 gN in the scoring pattern. An EAA index was calculated according to Oser (1959) using the AA composition of the whole egg protein standard published by Hidvegi and Bekes (1984). The EAA scores were calculated using the following formula:

Essential amino acid score (EAAS)=amino acid content in test protein (mg ? g-1)/(essential amino acid content in the FAO/WHO (1991) reference protein(mg ? g-1) The reference patterns were those established by FAO & WHO (FAO/WHO, protein quality evaluation, 1991) was used. Each data set along with the reference patterns was used to calculate the EAA index (EAAI). The EAAI was the geometric mean of scores of individual AAs and was equal to the antilogarithms of the individual scores. The individual scores were truncated to 100 if their values exceeded 100. Because tryptophan was destroyed by acid hydrolysis, it was excluded at 0 protein when calculating the AA score and EAAI.

Data analysis

The data reported in this study were the means of triplicate measurements. The means were compared by one-way analysis of variance (ANOVA) using SAS software (v8.2, SAS Institute Inc., Cary, NC, USA).

Fig. 4 'DN47' and 'NIL-DN47-Δα' seeds at different development stagesA, 'NIL-DN47-Δα' seeds; B, SDS-PAGE showing accumulation patterns of 'DN47' and 'NIL-DN47-Δα' in whole seeds extracted at various stages of maturation; D, Days after flowering.

Results

Evaluation of AA contents and nutritional quality of NIL-DN47-Δα seeds

As shown in Tables 1 and 2 the contents of crude proteins, the total AA, the total EAA and sulfurcontaining AAs (Met and Cys) in the NIL-DN47-Δαline increased by 4.11%, 4.16%, 5.20% and 11.96% as compared to the DN47, respectively. The concentrations of Thr, Val, Met and Ile increased significantly in thecgy-2 mutants, resulting in a significant increase of the total essential AA content. The concentrations of the sulfur-containing Met and Cys significantly increased in NIL-DN47-Δαbecause of promoted accumulation of Met. The total AA concentration also increased in NIL-DN47-Δαbecause of the enhanced contents of most AAs (Table 2).

Table 2 Comparison of amino acid and free amino acid content of mature seeds between 'DN47' and its near isogenic line 'NIL-DN47-Δα'

The increased content of the FAAs in NIL-DN47-Δαwas most pronounced for the Arg, which was more than two-fold as much as that in the DN47. In NIL-DN47-Δα, Arg constituted 36.81% of the FAAs, whereas the Asp and the Glu accounted for 11.63% and 9.32% of FAAs, respectively. The remaining 42.24% comprised other FAAs. The His content also showed two-fold increase in NIL-DN47-Δαbut remained much lower than Arg.

The general and significant increase in the contents of most FAAs resulted in a significant increase in contents of the total essential FAAs and the total FAAs (Table 2). The AA score was calculated according to the scoring pattern suggested by the FAO/WHO (FAO/WHO Protein quality evaluation, 1991). Both the total EAA content and the EAAI in NIL-DN47-Δαwere higher than those in the DN47 (Table 3). The EAAI values were assigned a maximum of 1.00 and a minimum of 0.01. Feedstuffs were rated as good quality protein sources when the EAAI was ≥0.90, adequate when it was about 0.80 or inadequate when it was below 0.70 (Oser, 1959). Our results suggested that the null-allele ofα-subunit positively affected the AA scores.

Table 3 Amino acid profile of mature seeds in 'DN47' and 'NIL-DN47-Δα'

Time course accumulation of FAAs in developing seeds

To study the dynamic changes contributing to the final concentrations of the FAAs in mature seeds, the levels and composition of the FAAs were quantified during seed development in the DN47 and NILDN47-Δα. The concentrations of the FAAs throughout 10 stages of seed development, from 15 to 60 DAF, were measured (Table 4, Fig. 5). Both temporal and rate differences were apparent (Figs. 5 and 6). The accumulation pattern of the total FAAs in NIL-DN47-Δαseeds was similar to that in the DN47 during the course of seed development (Fig. 5). However, the total FAA content in the DN47 was higher than that in NIL-DN47-Δαat early stages of development (15 and 20 DAF), both increased to maximum values at 25 DAF and then decreased proportionately throughout the maturation period (25 to 60 DAF). At 25 DAF, the contents of the total FAAs, the total free EAAs and the total sulfur-containing FAAs in NIL-DN47-Δαwere all higher than those in the DN47 (Table 4,Fig. 5). The major contributor to the difference in the FAAs content was the general increase in the contents of individual FAAs in NIL-DN47-Δα(Table 4) that exceeded the corresponding increases in the DN47. As from 25 to 60 DAF, the total FAAs of the DN47 and NIL-DN47-Δαdecreased from 9.3276 to 1.9867 mg ? g-1and from 10.8900 to 2.8267 mg ? g-1, respectively(Table 4). Throughout the later stages (25 to 60 DAF), the total FAA content of NIL-DN47-Δαwas higher than that of the DN47 at the corresponding developmental stages (Fig. 5). Accordingly, the elevated concentration of FAAs in thecgy-2 mutant seeds was a consequence of the promoted synthesis and/or accumulation of individual FAA during seed development. The accumulation patterns for each FAA during seed development (15 to 60 DAF) are summarized in Fig. 6.

Table 4 Comparison of free amino acid profiles in developing seeds of 'DN47' and 'NIL-DN47-Δα' from 15-60 days after flowering (DAF) (mg ? g-1)

Continued

Fig. 5 Comparison of free amino acid composition of 'DN47' and 'NIL-DN47-Δα' whole seed extracts at various stages of seed development 15-60 days after flowering (DAF)Individual values are average of three independent extractions and measurements.

FAA contents of soybean lines with different subunit deficiencies

To test the hypothesis that thecgy-2 mutation was responsible for the enhanced FAA contents, the FAA contents of the mature seeds of six additional strains were determined, which contained thecgy-2 gene in different genetic backgrounds, and the data to that of NIL-DN47-Δαwere compared.

The results of SDS-PAGE (Fig. 2) demonstrated the differences in protein composition of the tested genotypes. The recurrent parent DN47 containd all glycinin andβ-conglycinin subunits (Fig 2, Lane 1), whereas the null line S1 lacked theα-subunit ofβ-conglycinin (Fig. 2, Lanes 3 and 4). The null line S2 was deficient for theα-subunit ofβ-conglycinin and glycinin-group I (Fig. 2, Lanes 5 and 6). The null line S3 lacked theα′-subunit ofβ-conglycinin (Fig. 2,Lanes 7 and 8). In the null line S4, theα′-subunit ofβ-conglycinin and glycinin-group I were missing (Fig. 2, Lanes 9 and 10). The null line S5 lacks bothα′ andα-subunits ofβ-conglycinin (Fig. 2, Lanes 11 and 12).The null line S6 was defective for both theα′-andα-subunits ofβ-conglycinin and glycinin-group I (Fig. 2, Lanes 13 and 14). Table 5 showed the FAA contents of the single (S1 and S3), double (S2, S4 and S5) and triplemutant (S6) soybean seeds from six different storage protein-deficient lines (Table 1). In spite of the absence of variousβ-conglycinin or glycinin subunits, the crude protein contents in all mutant lines were significantly higher than those in the DN47 control (Table 1). Large differences in FAAs among the different genotypes were observed. Four mutant lines (S1, S2, S5 and S6) containing the cgy-2 allele exhibited higher levels of the FAAs compared with DN47 (Table 5, Fig. 7). The increase in the contents of Met and Cys resulted in elevated contents of sulfur-containing AAs (Met and Cys) in all of the six mutant lines. Out of the studied mutant lines, S2 (nullα, group I) had the highest contents of free Arg and total FAAs (Table 5).

Fig. 6 Comparison of free amino acids profiles from the whole seed extracts at various stages of seed development 15-60 days after flowering (DAF)

Table 5 Free amino acid composition (mg per g of dry weight) of mature seeds from 'DN47' and six soybean mutant lines lacking various β-conglycinin and glycinin subunits (mg ? g-1)

It was noteworthy that the free Arg contents in the six mutants were higher than those in DN47 (Table 5,Fig. 7). The significant increase in free Arg content affected greatly the final total FAA contents.

The free Arg contents of the mutants ranged from 1.7877 to 8.4473 mg ? g-1(Table 5), with significant differences between the mutant lines. The single null genotypes S1 (nullα) and S3 (nullα′) showed higher free Arg and the total FAA contents than DN47. No cumulative increase in the FAA content occurred in the double-null S5 mutant (nullα,α′).The concentration of the FAAs in S5 (nullα,α′) was only slightly higher than that in DN47 but was similar to that of the single-null-allele mutants S1 and S3. These results indicated that the increase in the FAA content did not correspond to the numbers of the null alleles. However, combining the null mutations with a glycinin-group I null additively increased the content of free Arg and the total FAAs (Table 5, Fig. 7), the levels of free Arg in mature seeds of S2 (nullα, group I),S4 (nullα′, group I) and S6 (nullα,α′ and group I) were five to seven times as much as these in the DN47 (Table 5, Fig. 7). Accordingly, the trend of increasing free Arg contents in the tested genotypes was normal soybean (DN47)＜β-conglycinin subunitnull type genotypes (S1, S3 and S5)＜β-conglycinin combined with glycinin subunit-null type genotypes (S2, S4 and S6).

Fig. 7 Free amino acid compositions of mature seeds from 'DN47' control and the six mutantsIndividual values are expressed in mg ? g-1 of dry weight, and are the average of three independent extractions and measurements. Absent subunits are presented beneath the figure. G, Glycinin-group I. * On the amino acid name: essential amino acids.

Discussion

Synthesis and accumulation of protein is one of the main metabolic events during seed development in soybean. Soybean seeds contain relatively low levels of the FAAs where the AAs are incorporated into storage proteins. Our data, along with recently reported studies (Songet al., 2014; Takahashiet al., 2003; Kitaet al., 2010), indicated that reduction in the amount of storage proteins was compensated for by activation of an alternative pathway of nitrogen assimilation, resulting in the accumulation of large amounts of the FAAs in soybean seeds. In particular, overproduction of free Arg seems to be a common biochemical route that compensates for the retarded assimilation of nitrogen intoβ-conglycinin and glycinin. Similar findings had been reported in several crops including maize (Zea mays) (Mertzet al., 1964; Mertz, 1997), barley (Hordeum vulgare) (Muncket al., 1970; Dollet al., 1974) and sorghum (sorghum bicolor)(Singh, 1973). In maize, the high-Lys opaque 2 mutant (Mertzet al., 1964; Mertz, 1997) had been reported to contain low levels of a Lys-poor seed storage protein (zein) and had compensatory increases in the Lys- and the Trp-rich non-zein seed proteins as well as free Lys and Trp as compared with the wild-type plants (Mertzet al., 1974; Misraet al., 1975). Many studies had attempted to explain the mechanism (s) leading to the high-FAA phenotype of opaque-2 maize (Mauriet al., 1993a, b; Colemanet al., 1995; Lopeset al., 1995; Burkhardtet al., 1979; Gillikinet al., 1997; Sunet al., 1997; Kimet al., 2004; Kimet al., 2006). The extent to which the Lys content of the opaque-2 mutant was elevated depended on the genetic background (Wang and Larkins, 2001). Commercial use of opaque-2 mutants became possible initially in Natal, South Africa (Geevers and Lake, 1992; Glover, 1992), when opaque-2-derived quality protein maize (QPM) lines with normal kernel properties and yields were generated. Since then, the QPM maize had been used worldwide.

In this study, significant variability was observed in the FAA contents of different soybean mutant lines. The lines derived from the BC1, namely S4, (nullα′, group I) and S6 (nullα,α′, group I), had significantly higher contents of Arg and the total FAAs than DN47. The lines derived from the BC3 generation plants, namely S1 (nullα), S3 (nullα′), S5 (nullα,α′) (Tables 1 and 5), S2 (nullα, group I) and F8 progenies derived from a three-way-cross (DN47/RiB//Suinong 10), showed the highest concentrations of free Arg and the total FAAs. These differences in the FAA contents may reflect variation in the genetic background. Performance evaluation trials conducted on storage protein subunit-null lines derived from single-crossed, backcrossed and three-way crossed lines (data not shown) showed similar free Arg and the FAA accumulation trends (three-Way-Cross＞BC1＞BC3＞single cross). Thus, it could be infered that differences between the mutant genotypes and DN47 were due to genotype-genetic background interactions, or interactions betweenβ-conglycinin and glycinin subunit-null genes.

L-Arginine is an important and unique amino acid in plants. It is important for nitrogen reserves and recycling as well as for biosynthesis of polyamines and nitric oxide. Polyamines and nitric oxide are important messengers that are involved in almost all physiological and biochemical processes, growth and development and adaptation of plants to stress. Arginine decarboxylase, arginase and nitric oxide synthase were the key enzymes in L-arginine catabolism in which polyamines were formedviathe arginine decarboxylase or arginine dehydrogenase pathways (Yang and Gao, 2007). Nitric oxide is formed through the nitric oxide synthase pathway. The relative activities of these three enzymes can control the direction of arginine metabolism. Arginine content is maintained at high levels in the roots during the overwintering periods. Arginine metabolism plays an important role in the perception and adaptation of plants to environmental disturbances. Arginine metabolism in developing soybean cotyledons had been intensively studied (Micallef and Shelp, 1989). The major N source for soybean seed germination had been shown to be Arg (Micallef and Shelp, 1989) and various studies on Arg metabolism had been reported (Glodraij and Polacco, 2000). However, little is known about the mechanisms by whichβ-conglycinin and glycinin subunit-deficiency mutations affect Arg biosynthesis. The studies were extended by developing variousβ-conglycinin and glycinin subunit-deficient single-gene-NILs that provided genetic materials for better understanding of underlying mechanisms. Furthermore, the combination of modern systembiological approaches, including transcriptomics, proteomics and metabolomics would elucidate how Arg pathways interact with regulatory networks that fine-tune the developmental-events in mutant soybean seeds.

The seeds of DN47 matured at about 60 DAF (Fig. 5)in which theαsubunit was clearly visible on the polyacrylamide gel electrophoresis (SDS-PAGE) at 30 DAF and then its band size rapidly increased from 35 DAF until maturity. Corresponding qRT-PCR analysis at each developmental stage revealed that theα-subunit gene began to show expression at 20 DAF. As of 25 DAF, there was exponential accumulation ofα-subunit gene transcripts (data not shown). These results were consistent with those of Meinkeet al., (1981) who found that the accumulation of 7Sα′ andα-subunits was initiated at 18-20 DAF, shortly after the termination of cell division in the developing cotyledons. Based on our data, it was concluded that the decrease of the FAAs from 25 DAF was accompanied by a substantial increase ofα-subunit gene expression. These data suggested that 25 DAF was an important turning point in the accumulation of the FAAs in soybean seeds.

Conclusions

The effects of the deficiency of the allergenicα-subunit on soybean AAs composition were investigated using the Dongnong 47 (DN47) genetic background. The contents of crude protein, the total AA, the total EAAs and sulfur-containing (Met and Cys) AAs increased by 4.11%, 4.16%, 5.20% and 11.96% in the NIL-DN47-Δαcompared with DN47, respectively. At the later stages of seed development (40-60 DAF), the total FAA contents of the NIL-DN47-Δαwere significantly higher than those of DN47. Interestingly, the increased synthesis of free Arg contributed greatly to the high FAA contents of various storage proteindeficient mutants. In addition, the increase of sulfurcontaining AAs in theα-null type NIL mainly resulted from raised Met contents. These data suggested that thecgy-2 allele ofβ-associated soy globulinα-subunit increased the seed protein content by increasing the contents of free amino acids, of which arginine and sulfur-containing amino acids were the main targets. This study provided theoretical basis for the mechanism by whichβ-conglycinin and glycinin subunit deficiency mutations regulate the AA contents in soybean seeds.