Mutations in the genes responsible for the synthesis of furan fatty acids resolve the light-induced off-odor in soybean oil.

Soybean oil is the second most produced edible vegetable oil and is used for many edible and industrial materials. Unfortunately, it has the disadvantage of 'reversion flavor' under photooxidative conditions, which produces an off-odor and decreases the quality of edible oil. Reversion flavor and off-odor are caused by minor fatty acids in the triacylglycerol of soybean oil known as furan fatty acids, which produce 3-methyl-2,4-nonanedione (3-MND) upon photooxidation. As a solution to this problem, a reduction in furan fatty acids leads to a decrease in 3-MND, resulting in a reduction in the off-odor induced by light exposure. However, there are no reports on the genes related to the biosynthesis of furan fatty acids in soybean oil. In this study, four mutant lines showing low or no furan fatty acid levels in soybean seeds were isolated from a soybean mutant library. Positional cloning experiments and homology search analysis identified two genes responsible for furan fatty acid biosynthesis in soybean: Glyma.20G201400 and Glyma.04G054100. Ectopic expression of both genes produced furan fatty acids in transgenic soybean hairy roots. The structure of these genes is different from that of the furan fatty acid biosynthetic genes in photosynthetic bacteria. Homologs of these two group of genes are widely conserved in the plant kingdom. The purified oil from the furan fatty acid mutant lines had lower amounts of 3-MND and reduced off-odor after light exposure, compared with oil from the wild-type.

Identification of a cytochrome P450 hydroxylase, CYP81E22, as a causative gene for the high sensitivity of soybean to herbicide bentazon.

KEY MESSAGE: A frame shift invoked by a single-base deletion in the gene encoding a cytochrome P450 hydroxylase, CYP81E22, causes the loss of bentazon detoxification function in soybean. Bentazon is an effective herbicide in soybean cultivation applied at post-emergence stages for control of several broadleaf weeds. However, some soybean cultivars are highly sensitive to bentazon and are killed upon application. In this study, the gene related to the high sensitivity of soybean cultivars to bentazon was mapped to chromosome 16, and its location was narrowed down to a 257-kb region where three cytochrome P450 genes were located. In these genes, a single-base deletion of cytosine was detected in the coding region of Glyma.16G149300, CYP81E22, at + 1465 bp downstream from the translation start codon, leading to a frame shift in the open reading frame and creating a premature stop codon. This stop codon resulted in the loss of more than half of the P450, and consequently, the remaining molecule failed to form a functioning protein. This single-base deletion was common among the highly sensitive cultivars screened from the soybean mini-core collection and other previously reported highly sensitive cultivars. Furthermore, we screened plant lines from the targeting-induced local lesions in genomes library of the soybean cultivar Enrei based on a modelled 3D structure of CYP81E22. The lines with mutations in Glyma.16G149300 were highly sensitive to bentazon, which provides strong evidence that Glyma.16G149300 is the gene responsible for high sensitivity to bentazon.

Single-base deletion in GmCHR5 increases the genistein-to-daidzein ratio in soybean seed.

Novel mutant alleles related to isoflavone content are useful for breeding programs to improve the disease resistance and nutritional content of soybean. However, identification of mutant alleles from high-density mutant libraries is expensive and time-consuming because soybean has a large, complicated genome. Here, we identified the gene responsible for increased genistein-to-daidzein ratio in seed of the mutant line F333ES017D9. For this purpose, we used a time- and cost-effective approach based on selective genotyping of a small number of F2 plants showing the mutant phenotype with nearest-neighboring-nucleotide substitution-high-resolution melting analysis markers, followed by alignment of short reads obtained by next-generation sequencing analysis with the identified locus. In the mutant line, GmCHR5 harbored a single-base deletion that caused a change in the substrate flow in the isoflavone biosynthetic pathway towards genistein. Mutated GmCHR5 was expressed at a lower level during seed development than wild-type GmCHR5. Ectopic overexpression of GmCHR5 increased the production of daidzein derivatives in both the wild-type and mutant plants. The present strategy will be useful for accelerating identification of mutant alleles responsible for traits of interest in agronomically important crops.

High-Throughput Screening and Characterization of a High-Density Soybean Mutant Library Elucidate the Biosynthesis Pathway of Triterpenoid Saponins.

Triterpenes (C30) constitute one of the diverse class of natural products with potential applications in food, cosmetic and pharmaceutical industries. Soyasaponins are oleanane-type triterpenoids widespread among legumes and particularly abundant in soybean seeds. They have associated with various pharmacological implications and undesirable taste properties of soybean-based food products. Uncovering the biosynthetic genes of soyasaponins will provide new opportunities to control the pathway for human benefits. However, the pathway of soyasaponin biosynthesis has not been fully elucidated in part because of a paucity of natural mutants. Here, we applied a structured high-density soybean mutant library for the forward genetic screening of triterpenoid biosynthesis. The seed soyasaponin polymorphism in the mutant library was evaluated using a high-throughput thin-layer chromatography and liquid chromatography tandem mass spectrometry analysis. This screening identified 35 mutants (3.85% of 909 mutant lines) with seven unusual soyasaponin phenotypes (Categories 1-7), which was greater than the number of natural mutants reported previously (22 mutants, 0.18% of ∼12,428 accessions). Nine unique intermediates of soyasaponin biosynthesis were identified and their chemical structures were estimated based on their MS/MS fragment patterns. Based on published information, 19 mutants could be associated with loss of function of four individual soyasaponin biosynthesis genes identified through expressed sequence tag mining or positional cloning, whereas the remaining 16 mutants were novel and may facilitate discovery of the unknown biosynthetic genes of soyasaponins. Our approach and library may help to identify new phenotype materials and causative genes associated with specialized metabolite production and other traits.

Identification and characterization of a major QTL underlying soybean isoflavone malonylglycitin content.

Isoflavones in soybean seeds are responsible for plant-microbe interactions and defend against pathogens, and are also beneficial to human health. We used two biparental populations and mini core collection of soybean germplasm to identify and validate QTLs underlying the content of isoflavone components. We identified a major QTL, qMGly_11, which regulates the content of malonylglycitin, on chromosome Gm11, in populations bred from parents with high, low, and null glycitein contents. qMGly_11 explained 44.5% of phenotypic variance in a population derived from a cross between 'Aokimame' (high) and 'Fukuyutaka' (low) and 79.9% of that in a population between 'Kumaji-1' (null) and 'Fukuyutaka' (low). The effect was observed only in the hypocotyl. We further confirmed the effect of qMGly_11 in a mini-core collection, where it explained 57.1% of the genetic diversity of glycitin production and 56.5% of malonylglycitin production. qMGly_11 increased the contents of glycitin and malonylglycitin at the expense of daidzin and malonyldaidzin in all analyzed populations. We discuss the gene responsible for this QTL and the availability of the null allele for metabolic engineering of soybean seed isoflavones.

Isolation and characterization of induced mutants in the gene associated with seed cadmium accumulation in soybean.

Food contamination by cadmium (Cd) is a serious threat to human health. Thus, it is imperative to prevent Cd accumulation in staple crops like soybean. The development of low Cd accumulating cultivars is an effective solution. To this end, it is essential to identify the gene(s) controlling seed Cd accumulation. Although Glyma.09G055600 (GmHMA3) seems to be associated with Cd accumulation in soybean, it has not been established if it is responsible for seed Cd accumulation. In the present study, the effect of GmHMA3 on seed Cd accumulation in soybean was validated using three independent GmHMA3 mutants isolated from an ethyl methanesulfonate-induced soybean mutant library. Each of mutant had an amino acid substitution in GmHMA3 and segregating progenies were developed by crossing the original cultivar with each of the three mutants. The relationship between these three mutations and seed Cd accumulation was investigated. While two of them significantly increased seed Cd accumulation corresponding to previous reports of a natural missense mutation in GmHMA3, the other slightly decreased seed Cd accumulation. Overall, these results indicate that GmHMA3 is responsible for seed Cd accumulation in soybean.

Metabolic switching of astringent and beneficial triterpenoid saponins in soybean is achieved by a loss-of-function mutation in cytochrome P450 72A69.

Triterpenoid saponins are major components of secondary metabolites in soybean seeds and are divided into two groups: group A saponins, and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponins. The aglycone moiety of group A saponins consists of soyasapogenol A (SA), which is an oxidized ß-amyrin product, and the aglycone moiety of the DDMP saponins consists of soyasapogenol B (SB). Group A saponins produce a bitter and astringent aftertaste in soy products, whereas DDMP saponins have known health benefits for humans. We completed map-based cloning and characterization of the gene Sg-5, which is responsible for SA biosynthesis. The naturally occurring sg-5 mutant lacks group A saponins and has a loss-of-function mutation (L164*) in Glyma15g39090, which encodes the cytochrome P450 enzyme, CYP72A69. An enzyme assay indicated the hydroxylase activity of recombinant CYP72A69 against SB, which also suggested the production of SA. Additionally, induced Glyma15g39090 mutants (R44* or S348P) lacked group A saponins similar to the sg-5 mutant, indicating that Glyma15g39090 corresponds to Sg-5. Endogenous levels of DDMP saponins were higher in the sg-5 mutant than in the wild-type lines due to the loss of the enzyme activity that converts SB to SA. Interestingly, the genomes of palaeopolyploid soybean and the closely related common bean carry multiple Sg-5 paralogs in a genomic region syntenic to the soybean Sg-5 region. However, SA did not accumulate in common bean samples, suggesting that Sg-5 activity evolved after gene duplication event(s). Our results demonstrate that metabolic switching of undesirable saponins with beneficial saponins can be achieved in soybean by disabling Sg-5.

Isolation and Characterization of the Soybean Sg-3 Gene that is Involved in Genetic Variation in Sugar Chain Composition at the C-3 Position in Soyasaponins.

Soyasaponins are specialized metabolites present in soybean seeds that affect the taste and quality of soy-based foods. The composition of the sugar chains attached to the aglycone moiety of soyasaponins is regulated by genetic loci such as sg-1, sg-3 and sg-4. Here, we report the cloning and characterization of the Sg-3 gene, which is responsible for conjugating the terminal (third) glucose (Glc) at the C-3 sugar chain of soyasaponins. The gene Glyma.10G104700 is disabled in the sg-3 cultivar, 'Mikuriya-ao', due to the deletion of genomic DNA that results in the absence of a terminal Glc residue on the C-3 sugar chain. Sg-3 encodes a putative glycosyltransferase (UGT91H9), and its predicted protein sequence has a high homology with that of the product of GmSGT3 (Glyma.08G181000; UGT91H4), which conjugates rhamnose (Rha) to the third position of the C-3 sugar chain in vitro. A recombinant Glyma.10G104700 protein could utilize UDP-Glc as a substrate to conjugate the third Glc to the C-3 sugar chain, and introducing a functional Glyma.10G104700 transgene into the mutant complemented the sg-3 phenotype. Conversely, induction of a premature stop codon mutation in Glyma.10G104700 (W270*) resulted in the sg-3 phenotype, suggesting that Glyma.10G104700 was Sg-3. The gmsgt3 (R339H) mutant failed to accumulate soyasaponins with the third Rha at the C-3 sugar chain, and the third Glc and Rha conjugations were both disabled in the sg-3 gmsgt3 double mutant. These results demonstrated that Sg-3 and GmSGT3 are non-redundantly involved in conjugation of the third Glc and Rha at the C-3 sugar chain of soyasaponins, respectively.

Molecular characterization of cytosolic cysteine synthase in Mimosa pudica.

In the cysteine and mimosine biosynthesis process, O-acetyl-L-serine (OAS) is the common substrate. In the presence of O-acetylserine (thiol) lyase (OASTL, cysteine synthase) the reaction of OAS with sulfide produces cysteine, while with 3-hydroxy-4-pyridone (3H4P) produces mimosine. The enzyme OASTL can either catalyze Cys synthesis or both Cys and mimosine. A cDNA for cytosolic OASTL was cloned from M. pudica for the first time containing 1,410 bp nucleotides. The purified protein product from overexpressed bacterial cells produced Cys only, but not mimosine, indicating it is Cys specific. Kinetic studies revealed that pH and temperature optima for Cys production were 6.5 and 50 °C, respectively. The measured Km, Kcat, and Kcat Km-1 values were 159 ± 21 µM, 33.56 s-1, and 211.07 mM-1s-1 for OAS and 252 ± 25 µM, 32.99 s-1, and 130.91 mM-1s-1 for Na2S according to the in vitro Cys assay. The Cy-OASTL of Mimosa pudica is specific to Cys production, although it contains sensory roles in sulfur assimilation and the reduction network in the intracellular environment of M. pudica.

Selection criteria for SNP loci to maximize robustness of high-resolution melting analysis for plant breeding.

DNA markers are useful for identifying genes and developing new genetic materials for breeding and genetic research. High-resolution melting (HRM) analysis can detect a single nucleotide polymorphism (SNP) in two polymerase chain reaction (PCR) fragments as a melting temperature (Tm) difference without additional experimental steps, such as gel electrophoresis. To design a method for developing reliable HRM markers that discriminate between homozygous alleles containing SNPs, we tested new evaluation indexes related to the thermodynamics of double-stranded DNA to find one that maximizes the difference in Tm values between PCR fragments. We found that differences in the change in Gibbs free energy (ΔG°) correlated with actual differences in Tm values. Optimization of the nearest neighboring nucleotide (NNN) of a SNP by nucleotide substitution in the primer and reducing the size of the PCR fragment both enlarged the actual differences in Tm. The genetic DNA markers we developed by NNN substitution, termed NNNs-HRM markers, could be precisely mapped within soybean chromosomes by linkage analysis. We developed a Perl script pipeline to enable the automatic design of a massive number of NNNs-HRM markers; these scripts are freely available and would be useful for practical breeding programs for other plant species.

High-resolution mapping and characterization of xa42, a resistance gene against multiple Xanthomonas oryzae pv. oryzae races in rice (Oryza sativa L.).

Improvement of resistance against rice bacterial blight (BB) disease is an important breeding strategy in breeding programs across the world, especially in Africa and southern Asia where BB is more prevalent. This report describes a high-resolution map and characterization of xa42 at XA42 locus, a rice BB resistance gene in XM14, a mutant line originating from IR24. The candidate gene region was narrowed down from 582 kb, which had been obtained in our previous study, to 57 kb. XM14 shows brown spots in its leaves like lesion mimic mutants. This line also shows a shorter stature than the original cultivar IR24. In XA42 gene segregating populations, homozygotes of xa42 allele were consistently resistant to the six Japanese Xanthomonas oryzae pv. oryzae races used for this study. They also showed brown spots and markedly short stature compared with the other genotypes, suggesting that xa42 gene exhibits pleiotropic effects.

Chromosomal Aneuploidy Improves the Brewing Characteristics of Sake Yeast.

The effect of chromosomal aneuploidy on the brewing characteristics of brewery yeasts has not been studied. Here we report that chromosomal aneuploidy in sake brewery yeast (Saccharomyces cerevisiae) leads to the development of favorable brewing characteristics. We found that pyruvate-underproducing sake yeast, which produces less off-flavor diacetyl, is aneuploid and trisomic for chromosomes XI and XIV. To confirm that this phenotype is due to aneuploidy, we obtained 45 haploids with various chromosomal additions and investigated their brewing profiles. A greater number of chromosomes correlated with a decrease in pyruvate production. Especially, sake yeast haploids with extra chromosomes in addition to chromosome XI produced less pyruvate than euploids. Mitochondrion-related metabolites and intracellular oxygen species in chromosome XI aneuploids were higher than those in euploids, and this effect was canceled in their "petite" strains, suggesting that an increase in chromosomes upregulated mitochondrial activity and decreased pyruvate levels. These findings suggested that an increase in chromosome number, including chromosome XI, in sake yeast haploids leads to pyruvate underproduction through the augmentation of mitochondrial activity. This is the first report proposing that aneuploidy in brewery yeasts improves their brewing profile.IMPORTANCE Chromosomal aneuploidy has not been evaluated in development of sake brewing yeast strains. This study shows the relationship between chromosomal aneuploidy and brewing characteristics of brewery yeast strains. High concentrations of pyruvate during sake storage give rise to α-acetolactate and, in turn, to high concentrations of diacetyl, which is considered an off-flavor. It was demonstrated that pyruvate-underproducing sake yeast is trisomic for chromosome XI and XIV. Furthermore, sake yeast haploids with extra chromosomes produced reduced levels of pyruvate and showed metabolic processes characteristic of increased mitochondrial activity. This novel discovery will enable the selection of favorable brewery yeasts by monitoring the copy numbers of specific chromosomes through a process that does not involve generation/use of genetically modified organisms.

Identification of quantitative trait loci for flowering time by a combination of restriction site-associated DNA sequencing and bulked segregant analysis in soybean.

Soybean (Glycine max) has a paleopolyploid genome, and many re-sequencing experiments to characterize soybean genotypes have been conducted using next-generation sequencing platforms. The accumulation of information about single nucleotide polymorphisms (SNPs) throughout the soybean genome has accelerated identification of genomic regions related to agronomically important traits through association studies. However, although many efficient mapping techniques that use next-generation sequencing are available, the number of practical approaches to identify genes/loci is still limited. In this study, we used a combination of restriction site-associated DNA sequencing (RAD-seq) and bulk segregant analysis (BSA) to identify quantitative trait locus (QTLs) for flowering time in a segregating population derived from a cross between Japanese soybean cultivars. Despite the homogeneous genetic background of the parents, over 7000 SNPs were identified and can be used to detect QTLs by RAD-seq BSA analysis. By comparing genotype frequency between early and late-flowering bulks from the F3 segregating population, we identified a QTL on Gm10, which corresponds to the previously identified E2 locus, and a QTL on Gm04, which is close to the E8 locus. Out of these SNPs, more than 2000 were easily converted to conventional DNA markers. Our approach would improve the efficiency of genetic mapping.

Confirmation of the pleiotropic control of leaflet shape and number of seeds per pod by the Ln gene in induced soybean mutants.

Most soybean cultivars possess broad leaflets; however, a recessive allele on the Ln locus is known to cause the alteration of broad to narrow leaflets. The recessive allele ln has also been considered to increase the number of seeds per pod (NSP) and has the potential to improve yield. Recently, Gm-JAG1 (Glyma20g25000), a gene controlling Ln, has been shown to complement leaf shape and silique length in Arabidopsis mutants. However, whether Gm-JAG1 is responsible for those traits in soybean is not yet known. In this study, we investigated the pleiotropic effect of soybean Ln gene on leaflet shape and NSP by using two independent soybean Gm-jag1 mutants and four ln near isogenic lines (NILs). The leaflet shape was evaluated using a leaf image analysis software, SmartLeaf, which was customized from SmartGrain. The leaflets of both the Gm-jag1 mutants were longer and narrower than those of the wild-type plants. Interestingly, the image analysis results clarified that the perimeter of the mutant leaflets did not change, although their leaflet area decreased. Furthermore, one mutant line with narrow leaflets showed significantly higher NSP than that in the wild (or Ln) genotype, indicating that soybean Ln gene pleiotropically controls leaflet shape and NSP.

Mutant selection in the self-incompatible plant radish (Raphanus sativus L. var. sativus) using two-step TILLING.

Radish (Raphanus sativus L. var. sativus), a widely cultivated root vegetable crop, possesses a large sink organ (the root), implying that photosynthetic activity in radish can be enhanced by altering both the source and sink capacity of the plant. However, since radish is a self-incompatible plant, improved mutation-breeding strategies are needed for this crop. TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful method used for reverse genetics. In this study, we developed a new TILLING strategy involving a two-step mutant selection process for mutagenized radish plants: the first selection is performed to identify a BC1M1 line, that is, progenies of M1 plants crossed with wild-type, and the second step is performed to identify BC1M1 individuals with mutations. We focused on Rubisco as a target, since Rubisco is the most abundant plant protein and a key photosynthetic enzyme. We found that the radish genome contains six RBCS genes and one pseudogene encoding small Rubisco subunits. We screened 955 EMS-induced BC1M1 lines using our newly developed TILLING strategy and obtained six mutant lines for the six RsRBCS genes, encoding proteins with four different types of amino acid substitutions. Finally, we selected a homozygous mutant and subjected it to physiological measurements.

Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing.

BACKGROUND: Functions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest. RESULTS: To increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions. CONCLUSIONS: A soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding.

GmCOL1a and GmCOL1b Function as Flowering Repressors in Soybean Under Long-Day Conditions.

CONSTANS (CO) has a central role in the photoperiod response mechanism in Arabidopsis. However, the functions of legume CO genes in controlling flowering remain unknown. Here, we analyze the expression patterns of E1, E2 and GmCOL1a/1b using near-isogenic lines (NILs), and we further analyze flowering-related genes in gmcol1b mutants and GmCOL1a-overexpressing plants. Our data showed that both E3 and E4 up-regulate E1 expression, with the effect of E3 on E1 being greater than the effect of E4 on E1. E2 was up-regulated by E3 and E4 but down-regulated by E1. GmCOL1a/1b were up-regulated by E1, E2, E3 and E4. Although the spatial and temporal patterns of GmCOL1a/1b expression were more similar to those of AtCOL2 than to those of AtCO, gmcol1b mutants flowered earlier than wild-type plants under long-day (LD) conditions, and the overexpression of GmCOL1a caused late flowering under LD or natural conditions. In addition, GmFT2a/5a, E1 and E2 were down-regulated in GmCOL1a-overexpressing plants under LD conditions. Because E1/2 influences the expression of GmCOL1a, and vice versa, we conclude that these genes may function as part of a negative feedback loop, and GmCOL1a/b genes may serve as suppressors in photoperiodic flowering in soybean under LD conditions.

Red/Far Red Light Controls Arbuscular Mycorrhizal Colonization via Jasmonic Acid and Strigolactone Signaling.

Establishment of a nitrogen-fixing symbiosis between legumes and rhizobia not only requires sufficient photosynthate, but also the sensing of the ratio of red to far red (R/FR) light. Here, we show that R/FR light sensing also positively influences the arbuscular mycorrhizal (AM) symbiosis of a legume and a non-legume through jasmonic acid (JA) and strigolactone (SL) signaling. The level of AM colonization in high R/FR light-grown tomato and Lotus japonicus significantly increased compared with that determined for low R/FR light-grown plants. Transcripts for JA-related genes were also elevated under high R/FR conditions. The root exudates derived from high R/FR light-grown plants contained more (+)-5-deoxystrigol, an AM-fungal hyphal branching inducer, than those from low R/FR light-grown plants. In summary, high R/FR light changes not only the levels of JA and SL synthesis, but also the composition of plant root exudates released into the rhizosphere, in this way augmenting the AM symbiosis.

Positional cloning and characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering.

The complex and coordinated regulation of flowering has high ecological and agricultural significance. The maturity locus E1 has a large impact on flowering time in soybean, but the molecular basis for the E1 locus is largely unknown. Through positional cloning, we delimited the E1 locus to a 17.4-kb region containing an intron-free gene (E1). The E1 protein contains a putative bipartite nuclear localization signal and a region distantly related to B3 domain. In the recessive allele, a nonsynonymous substitution occurred in the putative nuclear localization signal, leading to the loss of localization specificity of the E1 protein and earlier flowering. The early-flowering phenotype was consistently observed in three ethylmethanesulfonate-induced mutants and two natural mutations that harbored a premature stop codon or a deletion of the entire E1 gene. E1 expression was significantly suppressed under short-day conditions and showed a bimodal diurnal pattern under long-day conditions, suggesting its response to photoperiod and its dominant effect induced by long day length. When a functional E1 gene was transformed into the early-flowering cultivar Kariyutaka with low E1 expression, transgenic plants carrying exogenous E1 displayed late flowering. Furthermore, the transcript abundance of E1 was negatively correlated with that of GmFT2a and GmFT5a, homologues of FLOWERING LOCUS T that promote flowering. These findings demonstrated the key role of E1 in repressing flowering and delaying maturity in soybean. The molecular identification of the maturity locus E1 will contribute to our understanding of the molecular mechanisms by which a short-day plant regulates flowering time and maturity.

Allelic variation of soybean flower color gene W4 encoding dihydroflavonol 4-reductase 2.

BACKGROUND: Flower color of soybean is primarily controlled by six genes, viz., W1, W2, W3, W4, Wm and Wp. This study was conducted to investigate the genetic and chemical basis of newly-identified flower color variants including two soybean mutant lines, 222-A-3 (near white flower) and E30-D-1 (light purple flower), a near-isogenic line (Clark-w4), flower color variants (T321 and T369) descended from the w4-mutable line and kw4 (near white flower, Glycine soja). RESULTS: Complementation tests revealed that the flower color of 222-A-3 and kw4 was controlled by the recessive allele (w4) of the W4 locus encoding dihydroflavonol 4-reductase 2 (DFR2). In 222-A-3, a single base was deleted in the first exon resulting in a truncated polypeptide consisting of 24 amino acids. In Clark-w4, base substitution of the first nucleotide of the fourth intron abolished the 5' splice site, resulting in the retention of the intron. The DFR2 gene of kw4 was not expressed. The above results suggest that complete loss-of-function of DFR2 gene leads to near white flowers. Light purple flower of E30-D-1 was controlled by a new allele at the W4 locus, w4-lp. The gene symbol was approved by the Soybean Genetics Committee. In E30-D-1, a single-base substitution changed an amino acid at position 39 from arginine to histidine. Pale flowers of T369 had higher expression levels of the DFR2 gene. These flower petals contained unique dihydroflavonols that have not yet been reported to occur in soybean and G. soja. CONCLUSIONS: Complete loss-of-function of DFR2 gene leads to near white flowers. A new allele of the W4 locus, w4-lp regulates light purple flowers. Single amino acid substitution was associated with light purple flowers. Flower petals of T369 had higher levels of DFR2 gene expression and contained unique dihydroflavonols that are absent in soybean and G. soja. Thus, mutants of the DFR2 gene have unique flavonoid compositions and display a wide variety of flower color patterns in soybean, from near white, light purple, dilute purple to pale.