Triterpenoid saponins are specialised metabolites distributed widely in the plant kingdom that consist of one or more sugar moieties attached to triterpenoid aglycones. Despite the widely accepted ...view that glycosylation is catalysed by UDP-dependent glycosyltransferase (UGT), the UGT which catalyses the transfer of the conserved glucuronic acid moiety at the C-3 position of glycyrrhizin and various soyasaponins has not been determined. Here, we report that a cellulose synthase superfamily-derived glycosyltransferase (CSyGT) catalyses 3-O-glucuronosylation of triterpenoid aglycones. Gene co-expression analyses of three legume species (Glycyrrhiza uralensis, Glycine max, and Lotus japonicus) reveal the involvement of CSyGTs in saponin biosynthesis, and we characterise CSyGTs in vivo using Saccharomyces cerevisiae. CSyGT mutants of L. japonicus do not accumulate soyasaponin, but the ectopic expression of endoplasmic reticulum membrane-localised CSyGTs in a L. japonicus mutant background successfully complement soyasaponin biosynthesis. Finally, we produced glycyrrhizin de novo in yeast, paving the way for sustainable production of high-value saponins.
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.
Plant uridine 5'-diphosphate glycosyltransferases (UGTs) influence the physiochemical properties of several classes of specialized metabolites including triterpenoids via glycosylation. To uncover ...the evolutionary past of UGTs of soyasaponins (a group of beneficial triterpene glycosides widespread among Leguminosae), the UGT gene superfamily in
,
,
,
and
genomes were systematically mined. A total of 834 nonredundant UGTs were identified and categorized into 98 putative orthologous loci (POLs) using tree-based and graph-based methods. Major key findings in this study were of, (i) 17 POLs represent potential catalysts for triterpene glycosylation in legumes, (ii) UGTs responsible for the addition of second (
: galactosyltransferase and
: arabinosyltransferase) and third (
: rhamnosyltransferase and
: glucosyltransferase) sugars of the C-3 sugar chain of soyasaponins were resulted from duplication events occurred before and after the hologalegina-millettoid split, respectively, and followed neofunctionalization in species-/ lineage-specific manner, and (iii) UGTs responsible for the C-22-
glycosylation of group A (arabinosyltransferase) and DDMP saponins (DDMPtransferase) and the second sugar of C-22 sugar chain of group A saponins (
: glucosyltransferase) may all share a common ancestor. Our findings showed a way to trace the evolutionary history of UGTs involved in specialized metabolism.
Significance Pod dehiscence is a critical step in the seed dispersal (shattering) of legume and crucifer crops and can cause significant yield losses. Upon drying, pod walls are dehisced by two ...factors: the reduction of pod-wall binding strength and the generation of dehiscing forces. Although the previously reported shattering-resistant mutants maintained binding strength, here, we show a gene regulating the dehiscing force. The gene, Pdh1 , encodes a dirigent family protein, known to be involved in lignification, which increases dehiscing forces by promoting torsion of dried pod walls. The loss-of-function pdh1 gene has been widely used as a shattering-resistance gene in soybean breeding. This knowledge could be useful in improving other legume and crucifer crops, as well as soybean breeding.
Pod dehiscence (shattering) is essential for the propagation of wild plant species bearing seeds in pods but is a major cause of yield loss in legume and crucifer crops. Although natural genetic variation in pod dehiscence has been, and will be, useful for plant breeding, little is known about the molecular genetic basis of shattering resistance in crops. Therefore, we performed map-based cloning to unveil a major quantitative trait locus (QTL) controlling pod dehiscence in soybean. Fine mapping and complementation testing revealed that the QTL encodes a dirigent-like protein, designated as Pdh1 . The gene for the shattering-resistant genotype, pdh1 , was defective, having a premature stop codon. The functional gene, Pdh1 , was highly expressed in the lignin-rich inner sclerenchyma of pod walls, especially at the stage of initiation in lignin deposition. Comparisons of near-isogenic lines indicated that Pdh1 promotes pod dehiscence by increasing the torsion of dried pod walls, which serves as a driving force for pod dehiscence under low humidity. A survey of soybean germplasm revealed that pdh1 was frequently detected in landraces from semiarid regions and has been extensively used for breeding in North America, the world’s leading soybean producer. These findings point to a new mechanism for pod dehiscence involving the dirigent protein family and suggest that pdh1 has played a crucial role in the global expansion of soybean cultivation. Furthermore, the orthologs of pdh1 , or genes with the same role, will possibly be useful for crop improvement.
Background and AimsThe timing of flowering has a direct impact on successful seed production in plants. Flowering of soybean (Glycine max) is controlled by several E loci, and previous studies ...identified the genes responsible for the flowering loci E1, E2, E3 and E4. However, natural variation in these genes has not been fully elucidated. The aims of this study were the identification of new alleles, establishment of allele diagnoses, examination of allelic combinations for adaptability, and analysis of the integrated effect of these loci on flowering.MethodsThe sequences of these genes and their flanking regions were determined for 39 accessions by primer walking. Systematic discrimination among alleles was performed using DNA markers. Genotypes at the E1–E4 loci were determined for 63 accessions covering several ecological types using DNA markers and sequencing, and flowering times of these accessions at three sowing times were recorded.Key ResultsA new allele with an insertion of a long interspersed nuclear element (LINE) at the promoter of the E1 locus (e1-re) was identified. Insertion and deletion of 36 bases in the eighth intron (E2-in and E2-dl) were observed at the E2 locus. Systematic discrimination among the alleles at the E1–E3 loci was achieved using PCR-based markers. Allelic combinations at the E1–E4 loci were found to be associated with ecological types, and about 62–66 % of variation of flowering time could be attributed to these loci.ConclusionsThe study advances understanding of the combined roles of the E1–E4 loci in flowering and geographic adaptation, and suggests the existence of unidentified genes for flowering in soybean,
Whole-genome re-sequencing is a powerful approach to detect gene variants, but it is expensive to analyse only the target genes. To circumvent this problem, we attempted to detect novel variants of ...flowering time-related genes and their homologues in soybean mini-core collection by target re-sequencing using AmpliSeq technology. The average depth of 382 amplicons targeting 29 genes was 1,237 with 99.85% of the sequence data mapped to the reference genome. Totally, 461 variants were detected, of which 150 sites were novel and not registered in dbSNP. Known and novel variants were detected in the classical maturity loci-E1, E2, E3, and E4. Additionally, large indel alleles, E1-nl and E3-tr, were successfully identified. Novel loss-of-function and missense variants were found in FT2a, MADS-box, WDR61, phytochromes, and two-component response regulators. The multiple regression analysis showed that four genes-E2, E3, Dt1, and two-component response regulator-can explain 51.1-52.3% of the variation in flowering time of the mini-core collection. Among them, the two-component response regulator with a premature stop codon is a novel gene that has not been reported as a soybean flowering time-related gene. These data suggest that the AmpliSeq technology is a powerful tool to identify novel alleles.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA ...introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of
Cas9
-free mutants lacking an allergenic gene,
Gly m Bd 30K
, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T
0
plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the
Cas9
gene in the T
1
generation, which included
Cas9
-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the
Cas9
-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T
0
plants, we could remove foreign DNA easily by genetic segregation in the T
1
generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.
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.
Triterpene saponins are a diverse group of biologically functional products in plants. Saponins usually are glycosylated, which gives rise to a wide diversity of structures and functions. In the ...group A saponins of soybean (Glycine max), differences in the terminal sugar species located on the C-22 sugar chain of an aglycone core, soyasapogenol A, were observed to be under genetic control. Further genetic analyses and mapping revealed that the structural diversity of glycosylation was determined by multiple alíeles of a single locus, Sg-1, and led to identification of a UDP-sugar-dependent glycosyltransferase gene (Glyma07g38460). Although their sequences are highly similar and both glycosylate the nonacetylated saponin A0-αg, the Sg-1 a allele encodes the xylosyltransferase UGT73F4, whereas Sg-1 b encodes the glucosyltransferase UGT73F2. Homology models and site-directed mutagenesis analyses showed that Ser-138 in Sg-1 a and Gly-138 in Sg-1 b proteins are crucial residues for their respective sugar donor specificities. Transgenic complementation tests followed by recombinant enzyme assays in vitro demonstrated that sg-1° is a loss-of-function alíele of Sg-1. Considering that the terminal sugar species in the group A saponins are responsible for the strong bitterness and astringent aftertastes of soybean seeds, our findings herein provide useful tools to improve commercial properties of soybean products.
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.
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.
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.
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