Niels C. Nielsen

TILLING to detect induced mutations in soybean

BackgroundSoybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the worlds protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU).ResultsWe screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (~1/140 kb), while another EMS population had a mutation density of ~1/250 kb. The remaining population had a mutation density of ~1/550 kb. Because of soybeans polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template.ConclusionThe development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.

The structure and complexity of the 11S polypeptides in soybeans

The 11S soybean proteins called glycinin are isolated as a 350,000 dalton complex that consists of six nonidentical subunits. Each subunit consists of an acidic polypeptide component linked to a basic component by a single disulfide bond. Initial translation products of glycinin subunits are single polypeptides of ca. 60,000 daltons that undergo both co- and posttranslational modification. The precursors have a short signal sequence, followed by the acidic component, a short linker polypeptide, the basic component and a short trailer peptide. Five major subunit types have been purified and characterized by amino acid sequence analysis. While all of them are clearly synthesized by a family of homologous genes, they can be separated into two groups based on sequence homologies. Group I subunits (A1aB2, A1bB1b, A12B1a) are uniform in size (Mr = 58,000), relatively rich in methionine, and exhibit ca. 90% sequence homology among members in the group. The group II subunits (A3 B4, A5A4B3) exhibit a similar level of homology among themselves, although they contain less methionine and are larger (Mr ″ 62,000–69,000) than group I subunits. Sequence homology between a member of one group and a member of the other is only 60–70%. Since the sulfur amino acid content of subunits is variable and genetic polymorphism in subunit composition has been documented, alteration of the functional and nutritional properties of these seed proteins by genetic manipulation may be possible.

Structural similarity between legumin and vicilin storage proteins from legumes

The primary structures for several members of both the vicilin and legumin families of storage proteins were examined using a computer routine based on amino acid physical characteristics. The comparison algorithm revealed that sequences from the two families could be aligned and share a number of predicted secondary structural features. The COOH‐terminal half of the subunits in both families displayed a highly conserved core region that was largely hydrophobic and in which a high proportion of the residues were predicted to be in beta‐sheet conformations. The central region of the molecules which contained mixed areas of predicted helical and sheet conformations showed more variability in residue selection than the COOH‐terminal regions. The NH2‐terminal segments of subunits from the two different families could not be aligned though they characteristically had a high proportion of residues predicted to be in helical conformations. The feature which most clearly distinguished subunits between the two families was an inserted span in the legumin group with a high proportion of acidic amino acids located between the central and COOH‐terminal domains. Residues in this insertion were predicted to exist mainly in helical conformation. Since considerable size variation occurs in this area amongst the legumin subunits, alterations in this region may have a minimal detrimental effect on the structure of the proteins.

A soybean G2 glycinin allergen. 2. Epitope mapping and three-dimensional modeling.

Background: Multiple allergens have been documented in soybean extracts. IgE from individuals allergic to soybeans, but not to peanut, has been shown by immunoblot analysis to bind to proteins with a molecular weight of approximately 22 kD. These findings suggested that this unique protein fraction from soybean might be responsible, in part, for soybean allergic reactivity. The objective of the present study was to characterize specific B cell epitopes, to determine if any amino acid was critical to IgE binding and to model the 22-kD G2 soybean allergen to the three-dimensional (3-D) phaseolin molecule. Methods: B cell epitopes were identified using SPOTs peptide analysis. Structural orientation of the IgE-binding regions was mapped to the 3-D phaseolin molecule using molecular modeling of the protein tertiary structure. Results: Eleven linear epitopes, representing 15 amino acid peptide sequences, bound to IgE in the glycinin molecule. These epitopes were predicted to be distributed asymmetrically on the surface of G2 trimers. Conclusions: Only 1 epitope could be rendered non-IgE binding by alanine substitutions in the peptide. The nonrandom distribution of the IgE binding sites provides new insight into their organization in trimers in 11S complexes of the G2 glycinin allergen.

Genetic alteration of soybean oil composition by a chemical mutagen

Soybean (Glycine max [L.] Merr. cv. Century) seeds were treated with ethylmethanesulfonate (EMS) and the M2 progeny were evaluated for fatty acid composition of the oil. Treatment with EMS significantly increased the variability in content of each of the fatty acids in comparison with those of the Century control. There was a strong inverse relationship between oleic and linoleic acids among seeds from M2 plants. This supports the hypothesis of sequential desaturation as the method of formation of unsaturated fatty acids in soybean oil. A genetically stable mutant with 3.4% linolenic acid was identified that was similar to the cultivar Century in days to maturity, plant height and resistance to lodging.

A soybean G2 glycinin allergen. 1. Identification and characterization.

Background: Multiple allergens have been documented in soybean extracts. IgE from individuals allergic to soybeans, but not to peanut, was shown by immunoblot analysis to bind to proteins with a molecular weight of approximately 21 kD. These findings suggested that unique proteins in soybeans might be responsible for soybean allergic reactivity. The objective of the present study was to identify unique proteins in soybean extracts that bind to specific IgE from soybean-sensitive individuals, and to characterize the allergen using physicochemical methods and IgE binding. Methods: Two-dimensional and preparative SDS-PAGE/IgE immunoblot analysis was used to identify a 22-kD soybean-specific allergen from crude soybean extracts. N-terminal sequence analysis was used to determine the identification of the protein binding IgE from soybean-sensitive individuals. Results: IgE immunoblot and amino acid sequence analysis identified the 22-kD protein as a member of the G2 glycinin soybean protein family. Further investigation revealed that the IgEs reacted with basic chains from each member of the glycinin family of soybean storage proteins. Conclusions: Each of the subunits from glycinin, the storage protein that is the most prevalent component of soybean, are major allergens.

Flavor improvement of soybean preparations by genetic removal of lipoxygenase-2

Lipoxygenase-mediated oxidation of fatty acids has long been implicated in the production of off-flavors in soybean products. This assumption was tested by use of preparations from soybean lines nearly isogenic to the cultivar Century that lack the lipoxygenase isozyme or isozyme combinations L1, L2, L3, L1 + L3, or L2 + L3. Near-isogenic lines were used to ensure that any effects observed were due to elimination of lipoxygenase isozymes, and not to other unrecognized genetic differences between lines. Full-fat soy flour and unblanched soymilk preparations from the lines were evaluated by a six-member taste panel for eight flavor and/or aroma attributes common to soybeans. By comparison to near-isogenic controls, removal of the L2 isozyme from soymilk preparations produced significantly lower scores for beany, rancid and oily flavor and aroma attributes, as well as higher scores for dairy and cereal flavor and aroma attributes. Similar trends were noted for soy flour flavor attributes. Thiobarbituric acid (TBA) numbers, a measure of lipid oxidation, were lower in homogenized soy flour suspensions from lines lacking L2. Removal of the L1 and L3 isozymes did not result in improved flavor scores or lower TBA numbers. Total oil content and the fatty acid profile of the near isolines did not vary appreciably. The results indicated that genetic removal of the L2 isozyme may reduce off-flavors in soy products.

Characterization of a null-allele for the Gy 4 glycinin gene from soybean*

SummaryA null allele for the Gy4 glycinin gene from the cultivar Raiden was sequenced and compared with a functional Gy4 gene from another cultivar. The results showed that the null phenotype probably resulted from a point mutation that changed the translation initiation codon from ATG to ATA. Transcripts of the mutant gene were detected in total RNA from seed, but the mRNAs did not become associated with polysomes as did functional Gy4 meassage in the control cultivar. This was probably due to premature dissociation of mutant gy4 mRNA from ribosomes due to nonsense codons during translation of an incorrect reading frame.

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

SummaryDNA 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 β-conglycinin. These data indicate that the glycinin gene family is more complex than previously thought.

The Biochemistry and Cell Biology of Embryo Storage Proteins

Seed legumes accumulate massive amounts of certain characteristic proteins. These include the 7S and US seed storage proteins, lectins and various 2S proteins. With the advent of molecular cloning techniques, a large and increasing number of primary structures of these proteins from many plants are available in the international databases. This review summarizes and compares sequences of the seed proteins presently available. To the extent that they are known, the genes which encode the proteins and mechanisms involved in their expression are summarized. Strategies involved in the assembly of the 7S and US protein oligomers are discussed, and data that relate existing storage protein to their evolutionary progenitors are described.