In Situ Raman Hyperspectral Analysis of Microbial Colonies for Secondary Metabolites Screening.

Since the discovery of penicillin, a vast array of microbial antibiotics has been identified and applied in the medical field. Globally, the search for drug candidates via microbial screening is ongoing. Traditional screening methods, however, are time-consuming and require labor-intensive sample processing, significantly reducing throughput. This research introduces a Raman spectroscopy-based screening system tailored to the in situ analysis of microbial colonies on solid culture media. Employing multivariate curve resolution-alternating least-squares (MCR-ALS) for spectral decomposition, our approach reveals the production of secondary metabolites at the single colony level. We enhanced the microbial culture method, enabling direct, high signal-to-noise (S/N) ratio Raman spectroscopic measurements of colonies of Escherichia coli and actinomycetes species. Through semisupervised MCR analysis using the known spectra of actinorhodin and undecylprodigiosin as references, we accurately assessed the production of these compounds by Streptomyces coelicolor A3(2). Furthermore, we herein successfully detected the production of amphotericin B by Streptomyces nodosus, even in the absence of prior spectral information. This demonstrates the potential of our technique in the discovery of secondary metabolites. In addition to enabling the detection of the above-mentioned compounds, this analysis revealed the heterogeneity of the spatial distribution of their production in each colony. Our technique makes a significant contribution to the advancement of microbial screening, offering a rapid, efficient alternative to conventional methods and opening avenues for secondary metabolites discovery.

Analysis of microbial dynamics in the soybean root-associated environments from community to single-cell levels.

Plant root-associated environments such as the rhizosphere, rhizoplane, and endosphere, are notably different from non-root-associated soil environments. However, the microbial dynamics in these spatially divided compartments remain unexplored. In this study, we propose a combinational analysis of single-cell genomics with 16S rRNA gene sequencing. This method enabled us to understand the entire soil microbiome and individual root-associated microorganisms. We applied this method to soybean microbiomes and revealed that their composition was different between the rhizoplane and rhizosphere in the early growth stages, but became more similar as growth progressed. In addition, a total of 610 medium- to high-quality single-amplified genomes (SAGs) were acquired, including plant growth-promoting rhizobacteria (PGPR) candidates while genomes with high GC content tended to be missed by SAGs. The whole-genome analyses of the SAGs suggested that rhizoplane-enriched Flavobacterium solubilizes organophosphate actively and Bacillus colonizes roots more efficiently. Single-cell genomics, together with 16S rRNA gene sequencing, enabled us to connect microbial taxonomy and function, and assess microorganisms at a strain resolution even in the complex soil microbiome.

Identification of Genes Responsible for the Synthesis of Glycitein Isoflavones in Soybean Seeds.

Soybean (Glycine max (L.) Merrill) isoflavones are among the most important secondary metabolites, with functional benefits for human health. Soybeans accumulate three aglycone forms of isoflavones: genistein, daidzein, and glycitein. Soybean landrace Kumachi-1 does not accumulate malonylglycitin at all. Gene structure analysis indicated that Glyma.11G108300 (F6H4) of Kumachi-1 has a 3.8-kbp insertion, resulting in a truncated flavonoid 6-hydroxylase (F6H) sequence compared to the wild-type sequence in Fukuyutaka. Mapping experiments using a mutant line (MUT1246) with a phenotype similar to that of Kumachi-1, with a single-nucleotide polymorphism (SNP) in F6H4, revealed co-segregation of this mutation and the absence of glycitein isoflavones. We also identified a mutant line (K01) that exhibited a change in the HPLC retention time of glycitein isoflavones, accumulating glycoside and malonylglycoside forms of 6-hydroxydaidzein. K01 contains an SNP that produces a premature stop codon in Glyma.01G004200 (IOMT3), a novel soybean isoflavone O-methyltransferase (IOMT) gene. We further analyzed transgenic hairy roots of soybeans expressing Glyma.11G108300 (F6H4) and Glyma.01G004200 (IOMT3). Those overexpressing F6H4 accumulated malonylglycoside forms of 6-hydroxydaidzein (M_6HD), and co-expression of F6H4 and IOMT3 increased the level of malonylglycitin but not of M_6HD. These results indicate that F6H4 and IOMT3 are responsible for glycitein biosynthesis in soybean seed hypocotyl.

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.

Development of a High-Quality/Yield Long-Read Sequencing-Adaptable DNA Extraction Method for Crop Seeds.

Genome sequencing is important for discovering critical genes in crops and improving crop breeding efficiency. Generally, fresh, young leaves are used for DNA extraction from plants. However, seeds, the storage form, are more efficient because they do not require cultivation and can be ground at room temperature. Yet, only a few DNA extraction kits or methods suitable for seeds have been developed to date. In this study, we introduced an improved (IMP) Boom method that is relatively low-cost, simple to operate, and yields high-quality DNA that can withstand long-read sequencing. The method successfully extracted approximately 8 µg of DNA per gram of seed weight from soybean seeds at an average concentration of 48.3 ng/µL, approximately 40-fold higher than that extracted from seeds using a common extraction method kit. The A260/280 and A260/230 values of the DNA were 1.90 and 2.43, respectively, which exceeded the respective quality thresholds of 1.8 and 2.0. The DNA also had a DNA integrity number value (indicating the degree of DNA degradation) of 8.1, higher than that obtained using the kit and cetyltrimethylammonium bromide methods. Furthermore, the DNA showed a read length N50 of 20.96 kbp and a maximum read length of 127.8 kbp upon long-read sequencing using the Oxford Nanopore sequencer, with both values being higher than those obtained using the other methods. DNA extracted from seeds using the IMP Boom method showed an increase in the percentage of the nuclear genome with a decrease in the relative ratio of chloroplast DNA. These results suggested that the proposed IMP Boom method can extract high-quality and high-concentration DNA that can be used for long-read sequencing, which cannot be achieved from plant seeds using other conventional DNA extraction methods. The IMP Boom method could also be adapted to crop seeds other than soybeans, such as pea, okra, maize, and sunflower. This improved method is expected to improve the efficiency of various crop-breeding operations, including seed variety determination, testing of genetically modified seeds, and marker-assisted selection.

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.

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.

Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica.

BACKGROUND: Intermediate Filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. OBJECTIVE: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. METHODS: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. RESULTS: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. CONCLUSION: Together with data from other histological studies, our results suggest that Djf- 1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress.

Domesticating Vigna Stipulacea: A Potential Legume Crop With Broad Resistance to Biotic Stresses.

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.

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.

Soybean antiviral immunity conferred by dsRNase targets the viral replication complex.

Eukaryotic positive-strand RNA viruses replicate their genomes in membranous compartments formed in a host cell, which sequesters the dsRNA replication intermediate from antiviral immune surveillance. Here, we find that soybean has developed a way to overcome this sequestration. We report the positional cloning of the broad-spectrum soybean mosaic virus resistance gene Rsv4, which encodes an RNase H family protein with dsRNA-degrading activity. An active-site mutant of Rsv4 is incapable of inhibiting virus multiplication and is associated with an active viral RNA polymerase complex in infected cells. These results suggest that Rsv4 enters the viral replication compartment and degrades viral dsRNA. Inspired by this model, we design three plant-gene-derived dsRNases that can inhibit the multiplication of the respective target viruses. These findings suggest a method for developing crops resistant to any target positive-strand RNA virus by fusion of endogenous host genes.

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 of novel MYB transcription factors involved in the isoflavone biosynthetic pathway by using the combination screening system with agroinfiltration and hairy root transformation.

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 (ß-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.

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.

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.

Genetic and functional characterization of Sg-4 glycosyltransferase involved in the formation of sugar chain structure at the C-3 position of soybean saponins.

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 ßa) 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 ßa 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 ßa 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.

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.

Cytosolic Cysteine Synthase Switch Cysteine and Mimosine Production in Leucaena leucocephala.

In higher plants, multiple copies of the cysteine synthase gene are present for cysteine biosynthesis. Some of these genes also have the potential to produce various kinds of ß-substitute alanine. In the present study, we cloned a 1275-bp cDNA for cytosolic O-acetylserine(thiol)lyase (cysteine synthase) (Cy-OASTL) from Leucaena leucocephala. The purified protein product showed a dual function of cysteine and mimosine synthesis. Kinetics studies showed pH optima of 7.5 and 8.0, while temperature optima of 40 and 35 °C, respectively, for cysteine and mimosine synthesis. The kinetic parameters such as apparent Km, kcat were determined for both cysteine and mimosine synthesis with substrates O-acetylserine (OAS) and Na2S or 3-hydroxy-4-pyridone (3H4P). From the in vitro results with the common substrate OAS, the apparent kcat for Cys production is over sixfold higher than mimosine synthesis and the apparent Km is 3.7 times lower, suggesting Cys synthesis is the favored pathway.

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.