Identification of a plant introduction soybean line with genetic lesions affecting two distinct glycinin subunits and evaluation of impacts on protein content and composition

Unlike other oilseeds, soybean (Glycine max [L.] Merr) is also valuable due to its direct conversion into human food. One notable example is the cheese-like product tofu. The quality of tofu is improved when protein subunits derived from two glycinin genes, Gy1 and Gy4, are reduced or absent. Here we report the discovery that one exotic soybean plant introduction line, PI 605781 B, has not only a previously described loss-of-expression mutation affecting one glycinin gene (gy4), but also bears an extremely rare, potentially unique, frameshift mutation in the Glycinin1 gene (gy1-a). We analyzed glycinin gene expression via qRT-PCR with mRNA from developing seeds, which revealed that the novel allele dramatically reduced Gy1 mRNA accumulation. Similarly, both A₄A₅B₃ and A_1aB_1a protein subunits were absent or at undetectable levels, as determined by two-dimensional protein fractionation. Despite the reduction in glycinin content, overall seed protein levels were unaffected. The novel gy1-a allele was found to be unique to PI 605871B in a sampling of 247 diverse germplasm lines drawn from a variety of geographic origins.     

Resolution and reconstitution of complex V of the mitochondrial oxidative phosphorylation system: properties and composition of the membrane sector

The antigenic properties of purified glycinin subunits were studied using antibodies prepared against them. Antisera against native glycinin did not react with the isolated subunits, and antibodies prepared against the purified subunits were not active against native glycinin. When native glycinin -was denatured, the antiglycinin immunoglobulins lost their ability to react with it, although the denatured complex was then recognized by antibodies against the purified subunits. Substantial structural rearrangement apparently occurred when the native complex was denatured and disaggregated. Acidic polypeptides A_1a, A_1b, and A₂ had similar determinants as judged by their reactions against A_1a and A_1a antisera. The reaction of the A₃ polypeptides with these antibodies was of lower intensity and in each case clear spurs of cross-reactivity were visible. No cross-reaction was detected between polypeptide A₄ and either anti-A_1a or A₂. Anti-A₃ antibodies reacted with each of the acidic polypeptides of glycinin, and distinct spurs of cross-reactivity were observed between A₃ vs A_1a, A₃ vs A₂, and A₃ vs A₄. B₁ Antisera developed a reaction of identity between basic polypeptides B₁ and B₂, but reacted very weakly with B₃ and B₄. The acidic and basic polypeptides of glycinin were immunologically unrelated. The results demonstrated that immunological tests would successfully differentiate some members of the family of acidic subunits, and other immunoglobulins would discriminate between members of the family of basic subunits.     

Polymorphism and Expression of cDNAs Encoding Glycinin Subunits

The nucleotide sequence of cDNA encoding the glycinin A₂B_1a subunit from var. Shirotsurunoko was determined and compared with that in the case of var. Bonminori. The comparison showed six nucleotide substitutions in the coding sequence, one of which results in one amino acid replacement, and three in the 3'-noncoding region. These differences indicate the occurrence of polymorphism of the glycinin A₂B_1a subunit gene between the cultivars. The present data together with the previous results indicating the polymorphism of the A_1aB_1b subunit gene [(Utsumi et al., J. Agric. Food Chem., 35, 210 (1987)] suggest that the polymorphism is a general property of glycinin subunit genes. The expression of cDNAs encoding the A₂B_1a and A_1aB_1b subunits was examined. The results obtained in both in vivo- and in vitro-expression experiments indicate that the resultant products were readily degraded.     

Identification and characterization of DNA clones encoding group-II glycinin subunits

Summary DNA clones that encode the group-II subunits of soybean glycinin were identified and compared with clones for group-I subunits. The group-I clones hybridize weakly to those from group-II at low stringency, but fail to hybridize with them at moderate or high stringency. The genes for the group-II subunits are contained in 13 and 9 kb EcoRI fragments of genomic DNA in cultivar CX635-1-1-1. These fragments contain genes for subunits A₅A₄B₃ and A₃B₄, respectively. The larger size of mature group-II subunits compared with group-I subunits is correlated with a larger sized mRNA. However, the gross arrangement of introns and exons within the group-II coding regions appears to be the same as for the genes which encode group-I subunits. Messenger RNA for both groups of glycinin subunits appear in the seed at the same developmental interval, and their appearance lags slightly behind that of mRNAs for the a/a subunits of -conglycinin. These data indicate that the glycinin gene family is more complex than previously thought.Abbreviations bp base pairs - kb kilobase pairs - SDS sodium dodecyl sulfate Cooperative research between USDA/ARS and the Indiana Agric. Expt. Station. This work was supported in part by grants from the USDA Competitive Grants Program and the American Soybean Association Research Foundation. This is Journal Paper No. 10,078 from the Purdue Agricultural Experiment Station     

A complete cDNA coding for the sequence of glycinin A2B1a subunit precursor

Analysis of the A₂B_1a subunit precursor, one of the A₂-subunit family of glycinin, the main storage protein of soybean, revealed that it is composed of a signal peptide segment (18 amino acids), the A₂ acidic polypeptide (282 amino acids), followed by the B_1a basic polypeptide (185 amino acids). There was overall 63% homology between this subunit complex and pea legumin, which is an analogous protein to glycinin. As this degree of homology is rather higher than that in the A₃B₄ subunit, one of the A₃ subunit family, it seems that the genes encoding the A₂ subunit family are phylogenetically more strongly related to the legumin genes.     

Effect of Soy Protein Subunit Composition on the Rheological Properties of Soymilk during Acidification

The effect of soy protein subunit composition on the acid-induced aggregation of soymilk was investigated by preparing soymilk from different soybean lines lacking specific glycinin and β-conglycinin subunits. Acid gelation was induced by glucono-δ-lactone (GDL) and analysis was done using diffusing wave spectroscopy and rheology. Aggregation occurred near pH 5.8 and the increase in radius corresponded to an increase in the elastic modulus measured by small deformation rheology. Diffusing wave spectroscopy was also employed to follow acid gelation, and data indicated that particle interactions start to occur at a higher pH than the pH of onset of gelation (corresponding to the start of the rapid increase in elastic modulus). The protein subunit composition significantly affected the development of structure during acidification. The onset of aggregation occurred at a higher pH for soymilk samples containing group IIb (the acidic subunit A₃) of glycinin, than for samples prepared from Harovinton (a commercial variety containing all subunits) or from genotypes null in glycinin. The gels made from lines containing group I (A₁, A₂) and group IIb (A₃) of glycinin resulted in stiffer acid gels compared to the lines containing only β-conglycinin. These results confirmed that the ratio of glycinin/β-conglycinin has a significant effect on gel structure, with an increase in glycinin causing an increase in gel stiffness. The type of glycinin subunits also affected the aggregation behavior of soymilk.     

Inheritance of alleles for Cgy1 and Gy4 storage protein genes in soybean

Summary A cultivar lacking the glycinin subunit A₅A₄B₃ (Raiden) was crossed with one lacking the -subunit of -conglycinin (Keburi). Analysis of F₂ and F₃ progeny indicated that the missing bands of the A₅A₄B₃ and the -subunit were each controlled by a recessive allele of two independently segregating genes. Gene symbols Gy ₄/gy ₄ and Cgy ₁/cgy ₁ were proposed for the genes which confer the presence or absence of the glycinin and conglycinin subunits, respectively.Cooperative research of USDA-ARS and the Indiana Agric. Exp. Stn., Purdue Univ., West Lafayette, IN 47907, USA. Indiana Agric. Exp. Stn. Journal Article 9675. Financial support from the American Soybean Association Research Foundation is gratefully acknowledged     

Characterization of a Gy4 glycinin gene from soybean Glycine max cv. Forrest

The glycinin gene family encoding the glycinin subunits in soybean plants is composed of at least five gene members. A genomic clone S312 containing the Gy4 gene from a genomic library of cv. Forrest was isolated and partially characterized. The organization of this gene was found to be similar to that of a null allele from cv. Raiden, but different from the Gy4 gene from cv. Dare. The complete nucleotide sequence of this gene has been determined. It is 2599 bp long consisting of four exons and three introns. Comparing the DNA sequences between this gene and the gene from Dare and a null allele from Raiden, the difference found in the coding region was 5-GCAGTGCAAG-3 (nt 824 to 833) in the former case versus 5-TGGAGTTGCAATT-3 (nt 1314 to 1326) in the latter case in the exon 2 domain, resulting in three amino acid differences and one amino acid absence. Some other differences were also found in the non-coding region. The coding sequence and 5-flanking region of the Gy4 gene, when compared with that of other legumin genes as well as group 1 glycinin subunit genes, revealed some interesting features: (1) a transposable element-like sequence was found in the hypervariable region (HVR) of the exon 3 domain, which was lacking in the legumin and the glycinin group 1 genes; (2) in the 5-flanking region from nt –145 to –1, two high-homology sequences were found: one from nt –141 to nt –132, the other from nt –118 to nt –92 which includes the legumin box and the RY repeat element.     

Genetic mapping of the Gy4 and Gy5 glycinin genes in soybean and the analysis of a variant of Gy4

The predominant storage protein of soybean [Glycine max (L.) Merr.] seed is a globulin called glycinin. Thus far five genes encoding glycinin subunits have been described, and these are denoted by the gene symbols Gy1 to Gy5. The objectives of this study were to map two of these genes, Gy4 and Gy5, and to conduct a genetic analysis of a subunit size-variant from an allele of Gy4. For this purpose a population was formed with an interspecific cross between PI 468916 (G. soja) and A81-356022 (G. max). The two size forms of G4, the subunit from Gy4, segregated codominantly in the mapping population, and were due to a short insertion in the hypervariable region of the mutant protein. The biochemical and molecular characteristics of the two subunits indicate that they are produced from alternate alleles of the same gene. The gene symbols Gy ^a and Gy ^b have been assigned to the normal and variant genes, respectively. When genomic DNA from the two parents was probed with a Gy4 cDNA, RFLPs were identified for both Gy4 and Gy5. Using these genetic markers, the Gy4 and Gy5 glycinin genes were mapped in linkage group O and F on the public soybean genomic map.Joint contribution of North Central Region, USDA-ARS and Journal Paper No. J-14736 of the Iowa Agric. and Home Economics Exp. Stn., Ames, IA 50011; Project 2763. This work was supported, in part, with grants from the Iowa State Biotechnology Program (No. 480-46-09) and the Iowa Soybean Promotion Board to RCS, and the American Soybean Association to NCN     

The glycinin box: a soybean embryo factor binding motif within the quantitative regulatory region of the 11S seed storage globulin promoter

The soybean embryo factor binding sequence in the glycinin A₂B_1a gene promoter was delimited to an A/T-rich 9 bp sequence, 5-TAATAATTT-3, designated as the glycinin box, by DNA footprinting and gel mobility shift assay using synthetic oligonucleotides. It was shown that the interaction with the factor takes place at a defined DNA sequence rather than at random A/T-rich sequence blocks in the glycinin 5 flanking region. There are four glycinin boxes in the quantitative regulatory region between positions – 545 and – 378 of the glycinin A₂B_1a promoter. Multiple nonamer motifs similar to the glycinin box were also found in the equivalent regions of other glycinin and legumin promoters, suggesting that they must be conserved as a binding site for the embryo factor that activates the differential and stage-specific expression of seed 11S globulin genes in leguminous plants.     

Molecular markers linked to genes affecting plant height in wheat using a doubled-haploid population

 Plant height in wheat (Triticum aestivum L. em Thell) is known to be under polygenic control. Crosses involving genes Rht-B1 and Rht-D1, located on chromosomes 4BS and 4DS, respectively, have shown that these genes have major effects. Two RFLP loci were found to be linked to these two genes (Xfba1-4B with Rht-B1 and Xfba211-4D with Rht-D1) by genotyping a population of F₁-derived doubled-haploid lines [‘Courtot’ (Rht-B1b+Rht-D1b)בChinese Spring’]. Using a well-covered molecular marker map, we detected three additional regions and one interaction influencing plant height. These regions, located on chromosome arms 4BS (near the locus Xglk556-4B), 7AL (near the locus Xglk478-7A) and 7BL (near the locus XksuD2-7B) explained between 5% and 20% of the variability for this trait in this cross. The influence of 2 loci from chromosome 4B (Xfba1-4B and Xglk556-4B) suggests that there could be a duplication of Rht-B1 on this chromosome originating from Cv ‘Courtot’. Moreover, an interaction effect between loci from chromosome arms 1AS (near the locus Xfba393-1A) and 1BL (near the locus Xcdo1188-1B) was comparable to or even higher than those of the Rht-B1b and Rht-D1b alleles. A model including the main effects of the loci from chromosomes 4B and 4D (Xfba1-4B, Xglk556-4B and Xfba211-4D) and the interaction effect between Xfba393-1A and Xcdo1188-1B is proposed, which explains about 50% of the variation in plant height. The present results are discussed in relation to those obtained using nullisomic or substitution lines. Received: 13 June 1997 / Accepted: 13 October 1997     

Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon

clpC ofBacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (P_A and P_B) were mapped upstream of the first gene. P_A resembles promoters recognized by the vegetative RNA polymerase Eσ^A. The other promoter (P_B) was shown to be dependent on σ^B, the general stress σ factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a σ^B-dependent manner. This is the first evidence that σ^B in B, subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by σ³² in Escherichia coli, indicating a new function of σ^B-dependent general stress proteins. P_B deviated from the consensus sequence of σ^B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the σ^B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the σ^A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream σ^A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.     

Analysis of the proposed Fe-SX binding region of Photosystem 1 by site directed mutation of PsaA in Chlamydomonas reinhardtii

The psaA and psaB genes of the chloroplast genome in oxygenic photosynthetic organisms code for the major peptides of the Photosystem 1 reaction center. A heterodimer of the two polypeptides PsaA and PsaB is thought to bind the reaction center chlorophyll, P700, and the early electron acceptors A₀, A₁ and Fe-S_X. Fe-S_X is a 4Fe4S center requiring 4 cysteine residues as ligands from the protein. As PsaA and PsaB have only three and two conserved cysteine residues respectively, it has been proposed by several groups that Fe-S_X is an unusual inter-peptide center liganded by two cysteines from each peptide. This hypothesis has been tested by site directed mutagenesis of PsaA residue C575 and the adjacent D576. The C575D mutant does not assemble Photosystem 1. The C575H mutant contains a photoxidisable chlorophyll with EPR properties of P700, but no other Photosystem 1 function has been detected. The D576L mutant assembles a modified Photosystem 1 in which the EPR properties of the Fe-S_A/B centers are altered. The results confirm the importance of the conserved cysteine motif region in Photosystem 1 structure.Dedicated to the memory of Daniel I. Arnon.     

Overexpression of <Emphasis Type="Italic">Arabidopsis</Emphasis> damaged DNA binding protein 1A (DDB1A) enhances UV tolerance

Damaged DNA Binding protein 1 (DDB1) is a conserved protein and a component of multiple cellular complexes. Arabidopsis has two homologues of DDB1: DDB1A and DDB1B. In this study we examine the role of DDB1A in Arabidopsis UV tolerance and DNA repair using a DDB1A null mutant (ddb1a) and overexpression lines. DDB1A overexpression lines showed higher levels of UV-resistance than wild-type in a range of assays as well as faster DNA repair. However a significant difference between wild-type plants and ddb1a mutants was only observed immediately following UV treatment in root length and photoproduct repair assays. DDB1A and DDB1B mRNA levels increased 3 h after UV exposure and DDB1A is required for UV regulation of DDB1B and DDB2 mRNA levels. In conclusion, while DDB1A is sufficient to increase Arabidopsis UV tolerance, it is only necessary for immediate response to UV damage.     

Overexpression of Rice Genes OsNRT1.1A and OsNRT1.1B Restores Chlorate Uptake and NRT2.1/NAR2.1 Expression in Arabidopsis thaliana chl1-5 Mutant

Nitrogen uptake by plants is a key step for efficient nitrogen use, which affects plant growth and yield. Arabidopsis thaliana gene NRT1.1 was identified as a transporter related to nitrate (NO₃⁻) signaling and uptake. In rice, three orthologs of NRT1.1, named OsNRT1.1A, OsNRT1.1B, and OsNRT1.1C, have been identified. This study evaluated the potential of OsNRT1.1A, OsNRT1.1B, and OsNRT1.1C in NO₃⁻ signaling and uptake through overexpression in the Arabidopsis chl1-5 mutant. The expression of OsNRT1.1A, OsNRT1.1B, and OsNRT1.1C was evaluated in the roots and shoots of rice cultivated with NO₃⁻ or NH₄⁺. OsNRT1.1A was expressed in the roots and shoots cultivated with NO₃⁻ and NH₄⁺. OsNRT1.1B was expressed predominantly in roots of rice cultivated with NO₃⁻, while the expression of OsNRT1.1C was low in roots and shoots. Arabidopsis chl1-5 plants were transformed by the floral dip method using Agrobacterium tumefaciens to overexpress OsNRT1.1A and the alternative splicing product named OsNRT1.1As, OsNRT1.1B, and OsNRT1.1C. The chlorate test showed the ability of OsNRT1.1A, OsNRT1.1B or OsNRT1.1C to take up chlorate, as evidenced by the decrease in fresh weight. The OsNRT1.1B lineages presented higher toxicity to chlorate. Gene expression analyses showed that the insertion of OsNRT1.1A and OsNRT1.1B into Arabidopsis chl1-5 induced the expression of NRT2.1 and NAR2.1. OsNRT1.1As overexpression did not significantly affect the expression of NRT2.1 and NAR2.1. The results show the differential ability of NRT1.1 orthologs in rice to take up chlorate and signal the expression of other nitrate transporters, which may affect the efficiency of nitrogen utilization and its uptake.

     

Adenosine A2B receptor antagonist suppresses differentiation to regulatory T cells without suppressing activation of T cells

Extracellular adenosine activates P1 receptors (A₁, A_2A, A_2B, A₃) on cellular membranes. Here, we investigated the involvement of P1 receptor-mediated signaling in differentiation to regulatory T cells (Treg). Treg were induced in vitro by incubating isolated CD4⁺CD62L⁺ naïve murine T cells under Treg-skewing conditions. Antagonists of A₁ and A_2B receptors suppressed the expression of Foxp3, a specific marker of Treg, and the production of IL-10, suggesting the involvement of A₁ and A_2B receptors in differentiation to Treg. We also investigated the effect of these antagonists on T cell activation, which is essential for differentiation to Treg, and found that A₁ antagonist, but not A_2B antagonist, suppressed T cell activation. We conclude that A₁ and A_2B receptors are both involved in differentiation to Treg, but through different mechanisms. Since A_2B antagonist blocked differentiation to Treg without suppressing T cell activation, it is possible that blockade of A_2B receptor would facilitate tumor immunity.     

Expression of the Stress-Related Genes for Glutathione S-Transferase and Ascorbate Peroxidase in the Most-Glycinin-Deficient Soybean Cultivar Tousan205 during Seed Maturation

Global analysis of gene expression profiles in most-glycinin-deficient cultivar Tousan205, was performed by DNA microarray analysis. It was confirmed that Tousan205 lacks mRNA expression of three glycinin subunit precursor genes, G1 (A1aB1x), G2 (A2B1a), and G5 (A3B4), and lacks G4 (A5A4B3) protein. Most glycinin subunits were deficient in mature seeds of Tousan205. We compared the gene expression of Tousan205 with those of parent cultivar, Tamahomare, which was used for crossbreeding of Tousan205. As a result, Tousan205 exhibited higher expression of some seed maturation proteins, and stress-related genes such as glutathione S-transferase and ascorbate peroxidase. This result indicates the possibility that the decrease of main storage protein, glycinin causes stress in soybean.