New sources of soybean seed meal and oil composition traits identified through TILLING

<p>Abstract</p> <p>Background</p> <p>Several techniques are available to study gene function, but many are less than ideal for soybean. Reverse genetics, a relatively new approach, can be utilized to identify novel mutations in candidate genes; this technique has not produced an allelic variant with a confirmed phenotype in soybean. Soybean raffinose synthase genes and microsomal omega-6 fatty acid desaturase genes were screened for novel alleles in mutagenized soybean populations.</p> <p>Results</p> <p>Four mutations in independent lines were identified in the raffinose synthase gene <it>RS2</it>; two mutations resulted in amino acid mutations and one resulted in an altered seed oligosaccharide phenotype. The resulting phenotype was an increase in seed sucrose levels as well as a decrease in both raffinose and stachyose seed oligosaccharide levels. Three mutations in independent lines were identified in the omega-6 fatty acid desaturase gene <it>FAD2-1A</it>; all three mutations resulted in missense amino acid mutations and one resulted in an altered seed fatty acid profile that led to an increase in oleic acid and a decrease in linoleic acid in the seed oil.</p> <p>Conclusion</p> <p>The oligosaccharide phenotype controlled by the novel <it>RS2 </it>allele is similar to previously observed seed oligosaccharide phenotypes in <it>RS2 </it>mutant (PI 200508) allele-containing lines. Due to the anti-nutritional characteristics of raffinose and stachyose, this represents a positive change in seed composition. The fatty acid phenotype controlled by the novel <it>FAD2-1A </it>allele controls an increase in oleic acid in the seed oil, a phenotype also observed in a line previously characterized to have a null allele of the <it>FAD2-1A </it>gene. Molecular marker assays were developed to reliably detect the inheritance of the mutant alleles and can be used in efficient breeding for these desired seed phenotypes. Our results serve as the first demonstration of the identification of soybean mutants controlling seed phenotypes discovered through the reverse genetics technique TILLING.</p

Composition, vigor, and proteome of mature soybean seeds developed under high temperature

The effects of high temperature treatment on soybean [Glycine max (L.) Merr.] seed composition, vigor, and proteome were investigated using mature dry seeds harvested from plants grown in environment-controlled chambers. High day/night temperatures (37/30[degrees]C) from stages R5 through R8 altered ratios of individual fatty acids to total fatty acid compared to the control (27/18[degrees]C). Concentration of sugars decreased, but total protein and phytic acid concentration were unchanged. High temperature resulted in a greater proportion of abnormal seeds, but normal-appearing seed exhibited reduced germination and vigor. Proteomic analysis detected 20 protein identities whose accumulations were changed by the high temperature. Fourteen spots were identified as seven subunits of seed storage proteins. The remaining six proteins were identified as those responding to abiotic stresses or having a function in respiration: (i) sucrose binding protein, (ii) Class III acidic endochitinase, (iii) heat shock protein (HSP22), (iv) late embryo abundant protein, (v) Bowman-Birk proteinase inhibitor, and (vi) formate dehydrogenase. High temperature during seed development changed soybean seed composition and decreased seed vigor, but also changed seed protein expression profiles

Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait

<p>Abstract</p> <p>Background</p> <p>The alteration of fatty acid profiles in soybean [<it>Glycine max </it>(L.) Merr.] to improve soybean oil quality is an important and evolving theme in soybean research to meet nutritional needs and industrial criteria in the modern market. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of the oil. Commodity soybean oil typically contains 20% oleic acid and the target for high oleic acid soybean oil is approximately 80% of the oil; previous conventional plant breeding research to raise the oleic acid level to just 50-60% of the oil was hindered by the genetic complexity and environmental instability of the trait. The objective of this work was to create the high oleic acid trait in soybeans by identifying and combining mutations in two delta-twelve fatty acid desaturase genes, <it>FAD2-1A </it>and <it>FAD2-1B</it>.</p> <p>Results</p> <p>Three polymorphisms found in the <it>FAD2-1B </it>alleles of two soybean lines resulted in missense mutations. For each of the two soybean lines, there was one unique amino acid change within a highly conserved region of the protein. The mutant <it>FAD2-1B </it>alleles were associated with an increase in oleic acid levels, although the <it>FAD2-1B </it>mutant alleles alone were not capable of producing a high oleic acid phenotype. When existing <it>FAD2-1A </it>mutations were combined with the novel mutant <it>FAD2-1B </it>alleles, a high oleic acid phenotype was recovered only for those lines which were homozygous for both of the mutant alleles.</p> <p>Conclusions</p> <p>We were able to produce conventional soybean lines with 80% oleic acid in the oil in two different ways, each requiring the contribution of only two genes. The high oleic acid soybean germplasm developed contained a desirable fatty acid profile, and it was stable in two production environments. The presumed causative sequence polymorphisms in the <it>FAD2-1B </it>alleles were developed into highly efficient molecular markers for tracking the mutant alleles. The resources described here for the creation of high oleic acid soybeans provide a framework to efficiently develop soybean varieties to meet changing market demands.</p

TILLING to detect induced mutations in soybean

<p>Abstract</p> <p>Background</p> <p>Soybean (<it>Glycine max </it>L. Merr.) is an important nitrogen-fixing crop that provides much of the world's 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).</p> <p>Results</p> <p>We 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 soybean's 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.</p> <p>Conclusion</p> <p>The 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.</p

Structures of Michaelis and Product Complexes of Plant Cytokinin Dehydrogenase: Implications for Flavoenzyme Catalysis

Cytokinins form a diverse class of compounds that are essential for plant growth. Cytokinin dehydrogenase has a major role in the control of the levels of these plant hormones by catalysing their irreversible oxidation. The crystal structure of Zea mays cytokinin dehydrogenase displays the same two-domain topology of the flavoenzymes of the vanillyl-alcohol oxidase family but its active site cannot be related to that of any other family member. The X-ray analysis reveals a bipartite architecture of the catalytic centre, which consists of a funnel-shaped region on the protein surface and an internal cavity lined by the flavin ring. A pore with diameter of about 4 Å connects the two active-site regions. Snapshots of two critical steps along the reaction cycle were obtained through the structural analysis of the complexes with a slowly reacting substrate and the reaction product, which correspond to the states immediately before (Michaelis complex) and after (product complex) oxidation has taken place. The substrate displays a “plug-into-socket” binding mode that seals the catalytic site and precisely positions the carbon atom undergoing oxidation in close contact with the reactive locus of the flavin. A polarising H-bond between the substrate amine group and an Asp-Glu pair may facilitate oxidation. Substrate to product conversion results in small atomic movements, which lead to a planar conformation of the reaction product allowing double-bond conjugation. These features in the mechanism of amine recognition and oxidation differ from those observed in other flavin-dependent amine oxidases.

Kinetic and chemical analyses of the cytokinin dehydrogenase-catalysed reaction: correlations with the crystal structure

CKX (cytokinin dehydrogenase) is a flavoprotein that cleaves cytokinins to adenine and the corresponding side-chain aldehyde using a quinone-type electron acceptor. In the present study, reactions of maize (Zea mays) CKX with five different substrates (N(6)-isopentenyladenine, trans-zeatin, kinetin, p-topolin and N-methyl-isopentenyladenine) were studied. By using stopped-flow analysis of the reductive half-reaction, spectral intermediates were observed indicative of the transient formation of a binary enzyme–product complex between the cytokinin imine and the reduced enzyme. The reduction rate was high for isoprenoid cytokinins that showed formation of a charge-transfer complex of reduced enzyme with bound cytokinin imine. For the other cytokinins, flavin reduction was slow and no charge-transfer intermediates were observed. The binary complex of reduced enzyme and imine product intermediate decays relatively slowly to form an unbound product, cytokinin imine, which accumulates in the reaction mixture. The imine product only very slowly hydrolyses to adenine and an aldehyde derived from the cytokinin N(6) side-chain. Mixing of the substrate-reduced enzyme with Cu(2+)/imidazole as an electron acceptor to monitor the oxidative half-reaction revealed a high rate of electron transfer for this type of electron acceptor when using N(6)-isopentenyladenine. The stability of the cytokinin imine products allowed their fragmentation analysis and structure assessment by Q-TOF (quadrupole–time-of-flight) MS/MS. Correlations of the kinetic data with the known crystal structure are discussed for reactions with different cytokinins