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A soybean [Glycine max (L.) Merr.] mutant line producing deep purple flowers (E013-C-1) was developed from an ethylmethane sulfonatetreated population of the cultivar 'Bay', which has purple flowers. Genetic analysis was performed on a cross between 'E013-C-1' and the cultivar 'Clark' that had purple flowers. F1 plants had purple flowers whereas F2 plants segregated into a 3:1 purple/deep purple ratio. The results suggest that a single gene controls flower color and that purple color is dominant to deep purple. F3 plants derived from F2 plants with deep purple flowers were fixed for deep purple flowers. F3 plants derived from F2 plants with purple flowers segregated into two families segregating for flower color and one family that was fixed for purple flower color. The results confirmed that a single gene controls flower color and its recessive allele is responsible for deep purple flower color. The gene was designated Wd. Linkage mapping with simple sequence repeat (SSR) markers suggested that the Wd gene was located between Satt612 and Sct_199 in chromosome 18. Deep purple petals contained 50% higher levels of anthocyanins than purple petals. The vacuolar pH of deep purple flowers was similar to that of purple ones. These results suggest that the Wd gene controls the amount of anthocyanins and it is responsible for the development of deep purple flowers. [ABSTRACT FROM AUTHOR]

A soybean line producing light purple flowers (E023-H-12) was developed from an ethyl methanesulfonate (EMS)-treated population of cultivar Bay. The objective of this study was to investigate the genetic and molecular basis of flower color variation in E023-H-12. Genetic analysis suggested that the W1 gene encoding a flavonoid 3'5'-hydroxylase (F3'5'H) controls light purple flower color. A single plant with purple flowers was generated in an F2 population derived from a cross between E023-H-12 and Clark-w1 with white flowers, probably because of intragenic recombination of the F3'5'H gene. The allele for light purple flower was designated as w1-lp2. The dominance relationships of the locus were W1 > w1-lp2 > w1. Flower petals of E023-H-12 had similar expression levels of the F3'5'H gene but they had 42% less anthocyanins compared with Bay. The lower anthocyanin content may account for the light purple color of this mutant line. The nucleotide sequence of the F3'5'H gene of E023-H-12 had a single nucleotide polymorphism (SNP) resulting in alteration of an amino acid (H137L). A derived cleaved amplified polymorphic sequence (dCAPS) marker to discriminate the SNP co-segregated with flower color. Thus, the amino acid substitution may be responsible for the lower anthocyanin content, and, consequently, light purple flower color. [ABSTRACT FROM AUTHOR]

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 upregulate 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 newlyidentified 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. [ABSTRACT FROM AUTHOR]

Abstract  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. [ABSTRACT FROM AUTHOR]

Auxin-binding protein (ABPI) is a putative receptor for auxin in the plasma membrane. We isolated a full-length cDNA encoding ABPI from radish by screening a cDNA library with its partial cDNA fragment generated by the reverse transcriptasepolymerase chain reaction (RT PCR) method. Radish abp1 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 Nlinked glycosylation signals. To analyze the biochemical properties of the radish abp1 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 abp1 product was glycosylated at both target sites in insect cells. [ABSTRACT FROM AUTHOR]