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In Lotus japonicus, quantitative trait loci (QTLs) for nitrogen fixation activity (acetylene reduction activity, ARA) and seed weight are located in the same region on chromosome 4 as the gene resp...

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.

Soybean isoflavones are functionally important secondary metabolites that are mainly accumulated in seeds. Their biosynthetic processes are regulated coordinately at the transcriptional level; however, screening systems for key transcription factors (TFs) are limited. Here we developed a combination screening system comprising a simple agroinfiltration assay and a robust hairy root transformation assay. First, we screened for candidate MYB TFs that could activate the promoters of the chalcone synthase (CHS) gene GmCHS8 and the isoflavone synthase (IFS) genes GmIFS1 and GmIFS2 in the isoflavone biosynthetic pathway. In the agroinfiltration assay, we co-transformed a LjUbi (Lotus japonicus polyubiquitin gene) promoter-fused MYB gene with target promoter-fused GUS (β-glucuronidase) gene constructs, and identified three genes (GmMYB102, GmMYB280, and GmMYB502) as candidate regulators of isoflavone biosynthesis. We then evaluated the functional regulatory role of identified three MYB genes in isoflavone biosynthesis using hairy roots transformation assay in soybean for the accumulation of isoflavones. Three candidate MYB genes showed an increased accumulation of total isoflavones in hairy root transgenic lines. Accumulation of total isoflavones in the three MYB-overexpressing lines was approximately 2-to 4-folds more than that in the vector control, confirming their possible role to regulate isoflavone biosynthesis. However, the significant accumulation of authentic GmCHS8, GmIFS1, and GmIFS2 transcripts could not be observed except for the GmMYB502-overexpressing line. Therefore, the analysis of isoflavone accumulation in transgenic hairy root was effective for evaluation of transactivation activity of MYB TFs for isoflavone biosynthetic genes. Our results demonstrate a simple and robust system that can potentially identify the function of orphan TFs in diverse plant metabolic pathways.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Soybean (Glycine max) oil normally contains 2%–4% stearic acid. Increased saturated fatty acid levels elevate the melting point of soybean oil and improve oxidative stability. Using X-ray irradiation, we isolated six high stearic acid mutants and investigated mutations in the gene encoding Δ9-stearoyl-ACP-desaturase (SACPD). Our analyses of the KK2 mutant revealed an approximately 155-kbp deletion in the GmSACPD-B region. We also identified a frameshift mutation in the KK24 mutant, corresponding with a base-pair deletion in the GmSACPD-C coding region. The other four mutants M2, M18, MM106, and MS827 also had 2.2–21.8-kbp deletions in the GmSACPD-C region. Thus, we designed co-dominant marker sets for KK2 and KK24 mutant alleles using multiplex polymerase chain reactions and the CEL I mismatch detection method, respectively. In addition, we quantified the degradation of GmSACPD-C mRNA with a single base-pair deletion and related it to nonsense-mediated mRNA decay and simultaneous reductions in GmSACPD-A and -B mRNA levels. This molecular mechanism may be exploited to introduce silent mutations in duplicate genes of soybean plants and other organisms.

Triterpenoid saponins are specialized metabolites, which are abundant in soybean seeds. They have a wide variety of effects on human health and physiology. The composition of sugar chain attached to the aglycone moiety of saponins can be controlled by genetic loci, such as Sg-1, 3, and 4. Among these, the homozygous recessive sg-4 impairs the accumulation of saponins that have an arabinose moiety at the second position of the C-3 sugar chain (i.e., saponins Ad and βa) in the hypocotyls. In this study, we found that sg-4 cultivars are disabled in Glyma.01G046300 expression in hypocotyls. This gene encodes a putative glycosyltransferase (UGT73P10) and is a homolog of GmSGT2 (UGT73P2) whose recombinant protein has been previously shown, in vitro, to conjugate the second galactose moiety at the C-3 position of soyasapogenol B monoglucuronide (SBMG). The sg-4 phenotype (absence of saponins Ad and βa in hypocotyls) was restored by introducing the Glyma.01G046300 genomic DNA fragment that was obtained from the Sg-4 cultivar 'Ibarakimame 7'. Although Glyma.01G046300 is expressed in the cotyledons even in the sg-4 cultivars such as 'Enrei', the induced premature stop codon mutation (W244*) resulted in impaired accumulation of saponin βa in this tissue also in the 'Enrei' genetic background. Furthermore, the recombinant Glyma.01G046300 protein was shown to conjugate the second Ara moiety at the C-3 position of SBMG using UDP-Ara as a sugar donor. These results demonstrate that Sg-4 is responsible for conjugation of the second Ara moiety at the C-3 position of soybean saponins.

Though crossing wild relatives to modern cultivars is a usual means to introduce alleles of stress tolerance, an alternative is de novo domesticating wild species that are already tolerant to various kinds of stresses. As a test case, we chose Vigna stipulacea Kuntze, which has fast growth, short vegetative stage, and broad resistance to pests and diseases. We developed an ethyl methanesulfonate-mutagenized population and obtained three mutants with reduced seed dormancy and one with reduced pod shattering. We crossed one of the mutants of less seed dormancy to the wild type and confirmed that the phenotype was inherited in a Mendelian manner. De novo assembly of V. stipulacea genome, and the following resequencing of the F2 progenies successfully identified a Single Nucleotide Polymorphism (SNP) associated with seed dormancy. By crossing and pyramiding the mutant phenotypes, we will be able to turn V. stipulacea into a crop which is yet primitive but can be cultivated without pesticides.

The barley hordoindoline genes (Hina and Hinb) are homologous to the wheat puroindoline genes (Pina and Pinb). These genes are involved in grain hardness, which is an important quality for barley processing. We identified novel variants of Hina and Hinb in 10 wild Hordeum species (H. bogdanii, H. brachyantherum, H. bulbosum, H. chilense, H. comosum, H. marinum, H. murinum, H. patagonicum, H. pusillum, and H. roshevitzii) covering all Hordeum genomes and preliminarily named them Hinc. These nucleotide sequences were highly similar to those of Puroindoline b-2 variant genes (Pinb-2v) and were located on chromosome 7I in H. chilense. The Hinc genes in H. bogdanii, H. bulbosum, H. patagonicum, and H. roshevitzii were pseudogenes possessing in-frame stop codons. We also found a partial Hinc sequence in H. murinum. This gene was not found in cultivated barley and H. vulgare subsp. spontaneum. The phylogenetic tree of Gsp-1, Hin, and Pin genes demonstrates that Hinc and Pinb-2v genes formed one cluster. Therefore, we considered that Hinc and Pinb-2v genes shared a common ancestral gene and were homologous to each other. We also studied the evolutional process of Gsp-1, Hin, and Pin genes. Our results suggested that Gsp-1 might be the most closely related to a putative ancestral gene on Ha locus.

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 β-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.

Mutant-based reverse genetics offers a powerful way to create novel mutant alleles at a selected locus. This approach makes it possible to directly identify plants that carry a specific modified gene from the nucleotide sequence data. Soybean [Glycine max (L.) Merr.] has a highly redundant paleopolyploid genome (approx. 1.1 Gb), which was completely sequenced in 2010. Using reverse genetics to support functional genomics studies designed to predict gene function would accelerate post-genomics research in soybean. Furthermore, the novel mutant alleles created by this approach would be useful genetic resources for improving various traits in soybean. A reverse genetic screening platform in soybean has been developed that combines more than 40,000 mutant lines with a high-throughput method, Targeting Local Lesions IN Genome (TILLING). In this review, the mutant-based reverse genetic approach based on this platform is described, and the likely evolution of this approach in the near future.

With 6 figures and 2 tables Abstract The mid-oleic-acid soybean mutant M23 has been frequently used to develop lines with elevated oleic acid content. M23, which was produced by X-ray irradiation, was previously shown to contain a large deletion including GmFAD2-1a, one of the genes encoding microsomal omega-6 fatty acid desaturase (GmFAD2). However, it was not possible then to develop a codominant marker for the selection of the M23 mutation because the extent of the deletion was unknown. Here, we report that PCR analysis of M23 using published soybean genome sequences as a reference revealed a 164 014-bp deletion including GmFAD2-1a and nineteen other predicted genes. We generated a codominant PCR-based marker that can distinguish wild-type/M23 heterozygotes from both homozygous classes. We genotyped F2 plants segregating for the M23 mutation and confirmed the association of genotype and phenotype. This information will be useful in developing new commercial cultivars of high-oleic-acid soybean. We discuss the possible functions of the genes in the deleted region of M23 in relation to reduced seed yield, which occurs frequently in soybean lines containing the M23 mutation.

The generation of useful mutant alleles of specific genes would accelerate conventional breeding programs in various commercially important crops. Common soybean oil is easily oxidized because it is rich in polyunsaturated fatty acids (PUFAs). Microsomal omega-6 fatty acid desaturase (FAD2), which introduces a second unsaturated bond into oleic acid, is a primary target for elevating oleic acid levels and reducing PUFA levels. The paleopolyploid soybean genome contains five FAD2 gene homologues, at least three of which (GmFAD2-1a, 2-1b, and 2-2a) are functional. In spite of their importance, very little genetic variation has been identified in these genes except in GmFAD2-1a, because fatty acid content is easily affected by environmental conditions such as temperature. Here we isolated novel mutant alleles of GmFAD2-1b from ethyl methanesulfonate-treated soybean mutant populations through Targeting Induced Local Lesions In Genomes (TILLING), a reverse genetic method. Evaluation of enzyme activity in a yeast heterologous expression system suggested that two mutant lines, ‘B12’ and ‘E11’, contain near-null and null alleles, respectively, of GmFAD2-1b. Furthermore, by combining GmFAD2-1a and GmFAD2-1b mutant alleles, we successfully generated soybean lines with >80% oleic acid content. TILLING could provide a practical method for expanding the genetic diversity of polyploid crops.

Elevating the oleic acid content of soybean (Glycine max (L.) Merr.) is a major focus of breeding programs. Previously, we created two high-oleic-acid soybean mutants, M23 and KK21, by X-irradiation. We expected them to have modifications in genes encoding microsomal omega-6 fatty acid desaturase. The objectives of this study were to evaluate which members of the GmFAD2 gene family contribute to oleic acid production during seed maturation, to characterize the mutant genes, and to establish molecular markers for breeding of high-oleic-acid soybeans. Three GmFAD2 genes were expressed in developing seeds; the gene products of GmFAD2-1a and GmFAD2-1b were more active than that of GmFAD2-2a during seed development. We identified different nucleotide modifications in GmFAD2-1a in M23 and KK21. Using nuclease-cleaved DNA fragment-length polymorphisms, we developed a novel molecular marker to distinguish between KK21 mutant and wild-type alleles. This information could be useful for improving soybean oil quality by using the mutant genes from M23 or KK21, and for screening novel high-oleic-acid soybean mutants.

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.

In order to keep the quality of vegetable oil stable, it is important to reduce the α-linolenic acid content. Previously, we developed three low-α-linonenic acid soybean mutants, ‘J18’, ‘M5’, and ‘M24’. In this study, we obtained and characterized four cDNAs, GmFAD3-1a , GmFAD3-1b , GmFAD3-2a , and GmFAD3-2b , encoding microsomal ω-3 fatty acid desaturase from soybean developing seeds. The GmFAD3-1b transcript does not accumulate in the ‘J18’ mutant, whereas ‘M5’ and ‘M24’ contain a 19-base pair and a single-base pair deletion in the coding region of the GmFAD3-1b and GmFAD3-1a genes, respectively. Furthermore, heterologous expression of these two mutant genes in Saccharomyces cerevisiae revealed that the COOH-terminal region of both mutant gene products is essential for their enzymatic activity. In this report, we also discuss the contribution of each microsomal ω-3 fatty acid desaturase gene for α-linolenic acid biosynthesis in soybean seeds.

Three soybean [Glycine max (L.) Merr.] germplasms were previously developed for unique fatty acid content: LPKKC-3 has fap1 and sop1 alleles for reduced palmitic acid, HPKKC-7 has fap2 and fapx alleles for elevated palmitic acid and DHL has the ol allele for elevated oleic acid, and fan and fanxa alleles for reduced linolenic acid. If these loci are independently inherited, soybean germplasms with useful combinations of these fatty acids could be developed that would result in a novel oil quality of great value for the food industry. Crosses were made between DHL and LPKKC-3, and DHL and HPKKC-7. The data from F2 seed and F2 progeny of DHL X LPKKC-3 indicated that the loci controlling reduced palmitic acid (fap1 and sop1) were independently inherited from the locus controlling elevated oleic acid (ol), and the loci controlling reduced linolenic acid (fan and fanxa). Thus, the germplasm (LPDHL) with 4.0% palmitic, 51.0% oleic and 2.9% linolenic acids was easily developed. The data from F2 seeds and F2 progeny of DHL X HPKKC-7 indicated that the loci controlling elevated palmitic acid (fap2 and fapx), and reduced linolenic acid (fan and fanxa) were independently inherited. The locus controlling elevated oleic acid (ol) was also distinctly segregated but the contents of oleic acid were found to be reduced due to the presence of loci for elevated palmitic acid. Thus, one germplasm (HPLL) with 22.5% palmitic acid and 2.7% linolenic acid, and another germplasm (MHPDHL) with 17.1% palmitic acid, 41.8% oleic and 2.9% linolenic acids were developed.

Microsomal omega-3 fatty acid desaturase is an essential enzyme in the production of the n-3 polyunsaturated fatty acid alpha-linolenic acid during the seed developing stage. We have constructed a chimeric gene consisting of a maize Ubi1-P-int and a soybean GmFAD3 cDNA, which was introduced into rice plants by Agrobacterium-mediated transformation. Ten transformants containing the chimeric gene were established and expression subsequently confirmed by Northern blotting. Furthermore, alpha-linolenic acid content of the T(1) seeds increased dramatically up to tenfold that of the control, and this phenotype was also stably inherited in the T(2) and T(3) progenies. These results demonstrate that the alpha-linolenic acid content of rice seed oil can easily be altered using the combination of a high-activity promoter and a GmFAD3 gene.

Soybean (Glycine max (L.) Merr.) oil typically contains 8% α-linolenic acid that is highly unstable and easily oxidized. This property is undesirable in many food and industrial applications. Genetic strategies for reducing α-linolenic acid content would enhance the commercial value. However, genetic resources for low α-linolenic acid content are limited among natural soybean variations. Microsomal omega-3-fatty acid desaturase (FAD3) is responsible for the synthesis of α-linolenic acid in the polyunsaturated fatty acid pathway. There are four FAD3 homologs (Glyma02g39230, Glyma11g27190, Glyma14g37350 and Glyma18g06950) in the soybean genome. While non-functional alleles have been reported for Glyma02g39230 (GmFAD3-1a) and Glyma14g37350 (GmFAD3-1b), little variation is seen in Glyma18g06950 (GmFAD3-2a). We isolated seven mutant GmFAD3-2a alleles, each containing a single-nucleotide substitution, from 39,100 independent mutant lines by using targeting induced local lesions in genomes (TILLING). Analysis of GmFAD3-2a transcripts and enzyme activities revealed that one missense mutant, ‘P1-A9’, contains a non-functional allele of GmFAD3-2a. By combining three non-functional alleles (GmFAD3-1a, GmFAD3-1b, and GmFAD3-2a), we generated soybean lines containing

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.

A high content of linolenic acid lcads to reduction of keeping quality and frying stability of cooking oil. The present study was conducted to obtain a further reduction of linolenic acid in soybean [Glycine max (L.) Merr.] oil by re-irradiation of M-5 and determine its genetic system. M-5 is a mutant with 4.5% Iinolenic acid content, derived from the cultivar Bay (8.0% Iinolenic acid). A seed lot from line M-5 was treated with X-ray irradiation and M2 plants were obtained from randomly selected seeds of Ml plants. The M2 plants were screened for reduced linolenic acid. One plant was found with 3.0% Iinolenic acid content and was named MS382. The M3 and M4 generations of this line proved that the character was fixed and significantly lower than the M-5 control. For inheritance studies, MS382 was reciprocally crossed with M-5(fan) and LOLL[fanfanxa, a recombinant of M-5 (fan)× M24 (fanxa)]. The F2 segregation ratio and the segregation of F3 seeds from F2 plants of MS382×M-5 indicated that reduced linolenic acid in MS382 was conbindly controlled by fan (M-5) and an additional gene. To determine if this additional gene was similar with the fanxa gene in LOLL, F2 seeds and F3 seeds from each F2 plant of MS382×LOLL were evaluated. No transgressive segregation for linolenic acid was found in this cross, indicating the genes for reduced linolenic acid content in MS382 and LOLL were identical. However, the mutant MS382 was developed by re-irradiation which indicates the practicability of this technique to develop new gene for further reduction of linolenic acid in soybean oil.

Extensive studies on the molecular mechanisms of vesicular trafficking have revealed that molecules involved in this cellular function are remarkably well conserved from yeast to higher plants. However, it is not clear at all how a variety of organisms maintain the individual divergent systems using the common machinery of vesicular traffic. We have been attempting to understand the roles and regulatory mechanisms of vesicular traffic in plants through the study of Rab/Ypt GTPases. Ara proteins are Rab/Ypt homologues ofArabidopsis, which are implicated in the regulation of vesicular traffic. Their biochemical properties are similar to those of the Rab/Ypt proteins from animal and yeast cells. The overexpression ofARA2 orARA4 causes pleiotropic morphological abnormalities in the transgenic tobacco plants. The GTPase cycle of Ara proteins has to be strictly controlled for their proper functions. We have identified two classes of regulator molecules of Ara2 and Ara4. One is the GTPase activating protein (GAP), and the other is the GDP dissociation inhibitor (GDI). GAP has been identified as an activity accelerating the hydrolysis of GTP by Ara2 or Ara4. GDI (AtGDI1) has been isolated as a molecule interacting with Ara4 using a novel method for detecting interactions between foreign molecules in yeast. Further studies on the interacting molecules should unveil the regulatory system of and signal transduction pathway via Ara proteins.

Auxin-binding protein (ABP1) is a putative receptor for auxin in the plasma membrane. We isolated a full-length cDNA encoding ABP1 from radish by screening a cDNA library with its partial cDNA fragment generated by the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Radish abpl mRNA was highly expressed in cotyledons, hooks and hypocotyls, and less so in roots of radish seedlings. The deduced amino acid sequence of radish abp1 consisted of conserved auxin-binding motifs, a signal peptide and ER-retention signal, and contained two potentially N-linked glycosylation signals. To analyze the biochemical properties of the radish abpl product, this cDNA was expressed in insect cells by the baculovirus vector system. The result of tunicamycin-treatment showed that the signal peptide was cleaved and that the radish abpl product was glycosylated at both target sites in insect cells.

Summary The cDNA fragments of auxin-binding protein (ABP1) were isolated by degenerated-primer mediated reverse transcription-polymerase chain reaction (RT-PCR) from dicotyledonous plant species, i.e. cress, mung bean, pea, radish and soybean, and monocotyledonous species, i.e. oat and rice. Cloned abp 1 cDNA fragments were sequenced and compared with known abp 1 clones from A. thaliana , maize, strawberry and tobacco at the amino acid level. In all plant species studied, the amino acid sequences of newly isolated abp 1 clones were highly conserved at two regions (region A: Thr-Pro-Ile-His-Arg-His-Ser-Cys-Glu-Glu-Ile/Val-Phe-Ile/Thr/Val-Val-Leu/Pro/Val-Lys-Gly-Xaa-Gly-Thr-LeuAy; region B: His-Glu-Asp-Leu-Gln-Phe/Val-Leu-Asp/Val-Ile/Val-Ile-Ser-Arg-Pro-Pro), which were previously reported to be important for auxin-binding. On the other hand, some putative residues of amino acids in the region between regions A and B could be found to be specific in dicot and monocot species, respectively. Southern-blot analysis indicated a small abp 1 gene family in all species studied. Northern-blot analysis indicated that the size of abp 1 mRNA transcripts in all species studied was conserved at approximately 850bp. The phyloge-netic tree of ABP1 was analyzed by the UPGMA method.

Summary ara genes encoding YPT/RAB like small GTP-binding proteins, which are involved in the vesicular transport system in various eukaryotic cells, have been isolated from Arabidopsis thaliana . To determine the subcellular localization of ARA-4 protein, suspensions of cultured transgenic tobacco cells were analyzed using monoclonal antibody to Arabidopsis thaliana ARA-4 protein. ARA-4 protein was found in the microsomal membrane fraction. Similar results were obtained with Arabidopsis thaliana cells. The ARA-4 protein was readily released by detergents, such as 0.5 % sodium deoxycholate and NP-40. The results of immuno-blot analysis with extracts of different organs show that the ARA-4 protein is present in most organs of Arabidopsis thaliana plants, e. g. root, stem, flower and young fruit, at the same level in all organs except for root, where the level is lower.

Summary Comparison of adenylate kinase activities in rice seedlings ( Oryza sativa L. cv. Yamahoushi) that had been grown in the absence or presence of NaCl indicated that NaCI apparently stimulated enzyme activities in roots of this salt-sensitive japonica rice cultivar. Such stimulation of enzyme activities is not limited to a specific portion of root tissues. Furthermore, NaCl-induced adenylate kinase activation was confirmed in the indica rice cultivar IR 28 susceptible to salinity-stress, but not in the NaCl-tolerant indica cultivar Nona Bokra. Thus in salt sensitive rice plants, adenylate kinase may play some role in the adenylate homeostatis at the early stages of rice seedling growth subjected to salt-stress.

Palmitic acid is the most abundant (approx. 11% of total fatty acids) saturated fatty acid in conventional soybean seed oil. Increasing the saturated acid content of soybean oil improves its oxidative stability and plasticity. We have developed three soybean mutants with high palmitic acid content by X-ray irradiation. In this study, we successfully identified the mutated sites of two of these high-palmitic-acid mutants, J10 and M22. PCR-based mutant analysis revealed that J10 has a 206,203-bp-long deletion that includes the GmKASIIA gene and 16 other predicted genes, and M22 has a 26-bp-long deletion in the sixth intron of GmKASIIB. The small deletion in M22 causes mis-splicing of GmKASIIB transcripts, which should result in nonfunctional products. In addition, we designed co-dominant marker sets for these mutant alleles and confirmed the association of genotypes and palmitic acid contents in F(2) seeds of J10 X M22. This information will be useful in breeding programs to develop novel soybean cultivars with improved palmitic acid content. However, in the third mutant, KK7, we found no polymorphism in either GmKASIIA or GmKASIIB, which suggests that several unknown genes in addition to GmKASIIA and GmKASIIB may be involved in elevating the palmitic acid content of soybean seed oil.

The coat protein (CP) gene derived from Turnip mosaic virus (TuMV) isolate JO was introduced into Arabidopsis thaliana and the resulting transgenic progenies were analyzed for resistance to TuMV. Transgenic Arabidopsis plants with no detectable transcripts of the introduced CP gene exhibited complete resistance to TuMV. There was no significant correlation between the resistance and the copy number of the transgene. Instead, small interfering RNAs (siRNAs) were detected in these resistant plants, indicating that the resistance is attributed to RNA silencing. The RNA-mediated resistance was not only inherited over successive generations but also effective against 17 worldwide TuMV isolates with different pathogenicity. Comparative analysis of the CP genes among the 17 TuMV isolates revealed that the 380-nt in the 3′ region is highly conserved, suggesting the importance of the 3′ conserved region for broad-spectrum resistance. These results indicate that introduction of the TuMV-CP gene into the target Brassicaceae plants followed by selecting transformants that show RNA silencing for the transgenes can be an effective and reliable strategy for developing crucifer crops with a broad spectrum of resistance to TuMV.

T-DNA binary vectors are often used in plant transformation experiments. Because they are usually very large and have few restriction sites suitable for DNA ligation reactions, cloning DNA fragments into these vectors is difficult. We provide herein an alternative to cloning DNA fragments into very large vectors. Our yeast-based recombineering method enables DNA fragments to be cloned into certain types of T-DNA binary vectors by one-step transformation without the requirement of specific recombination sites or precisely positioned restriction ends, thus making the cloning process more flexible. Moreover, this method is inexpensive and is applicable to multifragment cloning.

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.

Two auxin-inhibiting substances were isolated from light-grown maize shoots. One was a new compound and determined from its spectral data as 4-Cl-6,7-dimethoxy-2-benzoxazolinone (Cl-DMBOA). Another was identified from its spectral data as a known compound, 6,7-dimethoxy-2-benzoxazolinone (DMBOA). Above concentrations of 10 −5 M, Cl-DMBOA inhibited the auxin-inducing elongation in the Avena coleoptile section test. Its inhibitory activity was almost comparable to that of DMBOA and higher than that of 6-methoxy-2-benzoxazolinone (MBOA) which we previously isolated.

The structure-activity relationship of lepidimoide and its analogues was investigated by means of the Amaranthus caudatus L. hypocotyl elongation test. In addition, the activities of alpha-D-galacturonic acid and L-(+)-rhamnose, which are component sugars of lepidimoide, were also studied. The carboxylic acid free type of lepidimoide showed growth-promoting activity as high as the original lepidimoide (sodium type). The acetylated compound showed considerably higher activity than lepidimoide, whereas the methylated lepidimoide did not show any activity. The hydroxylated lepidimoide without a double bond in the C-4,5 position showed lower activity. The sugar alcohol type of lepidimoide [2-O-(alpha-D-glucopyranosyl)-L-rhamnose] showed the highest activity in all the compounds studied. alpha-D-Galacturonic acid, L-(+)-rhamnose and their mixtures, which are component sugars of lepidimoide, exhibited only slight or no activity, respectively. D-Glucose and the mixture of D-glucose and L-(+)-rhamnose were also slightly active or inactive. These data suggest that the active sites in the chemical structure of the lepidimoide are the uronic acid derivative bearing an alpha,beta-unsaturated carboxylate bonded to rhamnose via an alpha-glucoside linkage and a double bond in the C-4,5 position in the uronic acid.

The occurrence in the plant kingdom of the allelopathic substance, lepidimoide, was studied. Lepidimoide is widespread in the exudates from all plant species studied. Lepidimoide occurred in especially large amounts in the exudates of germinated seeds of sunflower and buckwheat, but it was also detected in those of rice, lettuce, slender smaranth, leek and persian speedwell. Lepidimoide amounts did not differ greatly among genera or families.

The Arabidopsis Ara proteins belong to the Rab/Ypt family of small GTPases, which are implicated in intracellular vesicular traffic. To understand their specific roles in the cell, it is imperative to identify molecules that regulate the GTPase cycle. Such molecules have been found and characterized in animals and yeasts but not in plants. Using a yeast system, we developed a novel method of functional screening to detect interactions between foreign genes and identified this Rab regulator in plants. We found that the expression of the ARA4 gene in yeast ypt mutants causes exaggeration of the mutant phenotype. By introducing an Arabidopsis cDNA library into the ypt1 mutant, we isolated a clone whose coexpression overcame the deleterious effect of ARA4. This gene encodes an Arabidopsis homolog of the Rab GDP dissociation inhibitor (GDI) and was named AtGDI1. The expression of AtGDI1 complemented the yeast sec19-1 (gdi1) mutation. AtGDI1 is expressed almost ubiquitously in Arabidopsis tissues. The method described here indicates the physiological interaction of two plant molecules, Ara4 and GDI, in yeast and should be applicable to other foreign genes.