DNA methylation in higher organisms has become an expanding field of study as it often involves the regulation of gene expression. Although Whole Genome Bisulfite Sequencing (WG-BS) based on next-generation sequencing (NGS) is the most versatile method, this is a costly technique that lacks in-depth analytic power. There are no conventional methods based on NGS that enable researchers to easily compare the level of DNA methylation from the practical number of samples handled in the laboratory. Although the targeted BS method based on Sanger sequencing is generally used in this case, it lacks in-depth analytic power. Therefore, we propose a new method that combines the high throughput analytic power of NGS and bioinformatics with the specificity and focus offered by PCR-amplification-based bisulfite sequencing methods. We use in silico size sieving of DNA-fragments and primer matchings instead of whole-fragment alignment in our bioinformatics analyses, and named our method SIMON (Simple Inference for Methylome based On NGS). The results of our targeted BS method based on NGS (SIMON method) show that small variations in DNA methylation patterns can be precisely and efficiently measured at a single nucleotide resolution. SIMON method combines pre-existing techniques to provide a cost-effective technique for in-depth studies that focus on pre-identified loci. It offers significant improvements with regard to workflow and the quality of the acquired DNA methylation information. Because of the high accuracy of the analysis, small variations of DNA methylation levels can be precisely determined even with large numbers of samples and loci.

While the ‘Micro-Tom’ TILLING mutant library is used for a wide range of purposes, including both basic research of gene function and breeding of commercial cultivars, genome-wide distribution and frequency of mutations have not yet been thoroughly elucidated on a population scale. In this study, we developed a 96-plex exome sequencing method to identify and analyze mutations within the TILLING mutants that were developed in the University of Tsukuba. First, an Illumina paired-end sequencing coupled with 96-plex exome capture resulted in the acquisition of an exome sequence dataset with an average read count of 5.6 million for the 95 mutants. Over 98% of the capture target region could be covered by the short reads with an averaged read depth of 12.8, which enabled us to identify single nucleotide polymorphisms and Indels in a genome-wide manner. By subtracting intra-cultivar DNA variations that are present between wild-type ‘Micro-Tom’ lines, we identified 241,391 mutation candidates in 95 mutant individuals. Of these, 64,319 and 6,480 mutations were expected to cause protein amino acid substitutions or premature stop codon, respectively. Based on the exome mutation dataset, a mutant line designated ‘TOMJPW601’ was found to carry a premature stop codon mutation (W261*) in a putative auxin influx carrier gene SlLAX1 (Solyc09G014380), consistent with our previous report of its curly leaf phenotype. Our results suggested that a population-scale mutation database developed by multiplexed exome sequencing could be used for in silico mutant screening, which in turn could contribute to both gene function research and breeding programs.

The application of useful genes from model plants to crops is an important step to verify its agricultural usefulness. SYNC1, an asparaginyl-tRNA synthetase gene, was previously identified through the Full-length cDNA Over-eXpressor gene (FOX gene-hunting system) of Arabidopsis cDNA by using super-growing root (SR) culture of Lotus corniculatus, and was suggested to have a potential in increasing some amino acid contents and plant biomass. To identify the functionality of SYNC1 gene in a typical legume crop soybean, the effects of its overexpression in transgenic plants to agricultural traits and free amino acid contents were evaluated. The transgenic soybean plants were produced from infected half-seed explants of 1 day old seedlings with the suspension of Agrobacterium tumefaciens harboring expression vector pB7WG2D-SYNC1. The transgenic plants that overexpressed SYNC1 gene had increased asparagine and lysine contents in matured seeds, and increased aspartate, lysine, alanine and histidine contents in germinated seeds. The changes in those free amino acid contents affected plant morphology and led to significant increase in plant length, number of branches and number of branch nodes as yield components of soybean. The transgenic plants also showed a tendency of higher number of pods, seeds and total seed weight per plant. These results showed that the overexpression of SYNC1 gene contributes on the increase of plant free amino acid contents and biomass, and this approach is expected to be applicable in other legumes, grain and forage 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.

α-tomatine and dehydrotomatine are steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves, and flowers of tomatoes (Solanum lycopersicum) and function as defensive compounds against pathogens and predators. The aglycones of α-tomatine and dehydrotomatine are tomatidine and dehydrotomatidine (5,6-dehydrogenated tomatidine), and tomatidine is derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5α reduction, and C3 reduction. Our previous studies (Lee et al. 2019) revealed that Sl3βHSD is involved in the three reactions except for C5α reduction, and in the present study, we aimed to elucidate the gene responsible for the C5α reduction step in the conversion of dehydrotomatidine to tomatidine. We characterized the two genes, SlS5αR1 and SlS5αR2, which show high homology with DET2, a brassinosteroid 5α reductase of Arabidopsis thaliana. The expression pattern of SlS5αR2 is similar to those of SGA biosynthetic genes, while SlS5αR1 is ubiquitously expressed, suggesting the involvement of SlS5αR2 in SGA biosynthesis. Biochemical analysis of the recombinant proteins revealed that both of SlS5αR1 and SlS5αR2 catalyze the reduction of tomatid-4-en-3-one at C5α to yield tomatid-3-one. Then, SlS5αR1- or SlS5αR2-knockout hairy roots were constructed using CRISPR/Cas9 mediated genome editing. In the SlS5αR2-knockout hairy roots, the α-tomatine level was significantly decreased and dehydrotomatine was accumulated. On the other hand, no change in the amount of α-tomatine was observed in the SlS5αR1-knockout hairy root. These results indicate that SlS5αR2 is responsible for the C5α reduction in α-tomatine biosynthesis and that SlS5αR1 does not significantly contribute to α-tomatine biosynthesis.

Asparaptine, a conjugate of L-arginine and asparagusic acid, was found in green asparagus (Asparagus officinalis) using ultrahigh-resolution metabolomics for sulfur-containing metabolites (S-metabolites), called S-omics. Asparaptine has been shown to inhibit the activity of angiotensin-converting enzyme. Larger amounts of this S-metabolite are therefore required for further analysis; however, there are limitations that asparagus is a perennial plant and its spears, wherein asparaptine accumulates, can be mainly harvested at the spring to summer season. In order to overcome these, we prepared a callus and suspension cell line from green asparagus. Untargeted metabolome analysis using liquid chromatography-tandem mass spectrometry was performed in the materials as well as spears and three calluses derived from wild type Asparagus. The analysis demonstrated that the amount of asparaptine in the callus derived from the green asparagus was more than the others per mg dry weight. The suspension cell line treated with methyljasmonate showed the induction of asparaptine, suggesting that the asparaptine production is modifiable under appropriate culture conditions. The described materials can be utilized for the production of asparaptine and in integrated metabolomics to study the biosynthesis of this S-metabolite, which is currently unknown.

Anther culture is a fast tool to obtain double haploid plant lines for breeding purposes. In rice, this procedure is commonly performed in two steps: i) induction of calli from anthers and ii) regeneration of plantlets from calli. It has been stated that genotype highly influences the anther culture efficiency, so the media used in each step should be optimized for each variety. In this study, we tested different media modifications of an efficient protocol optimized for a medium sized grain temperate japonica NRVC980385, used as a control, in a long grain temperate japonica rice variety (NRVC20120346), and two long grain tropical japonica varieties (303012 and 303013).

We found that the addition of 150 mg l⁻¹ colchicine to the induction medium worked best for all genotypes except for NRVC20120346, whose best induction was obtained with the colchicine-free medium. Referring to regeneration, increased gelling agent in the medium provided the best rates in NRVC980385, improving our former NRVC980385-optimized anther culture protocol. Sorbitol fortified regeneration medium worked the best in the case of the long grain varieties. The presence of colchicine in the induction medium was also related to a higher obtention of double haploid plantlets. This study highlights that genotype is a key factor in the performance of rice anther culture. It has set a first anther culture study on long grain japonica varieties and optimizes the anther culture protocol for temperate japonica medium grain NRVC980385 with the use of colchicine and other additives that increase osmotic stress.

Reactive oxygen species generation is one of the most popular index of plant immune responses. Leaf disk assay has been commonly used for MAMP/elicitor-induced ROS analysis by many groups. However, the reproducibility of the leaf disk assay relies on the skills of the people engaged in the experiments and the experiment itself seems not suitable for some plant species, which had a tough leaf structure and lower penetration efficiency of MAMPs/elicitors. In this study, we prepared a handmade leaf cutter to cut out the leaf fragments with uniform size and slits. The use of such fragments obtained by the new leaf cutter as well as the increase of the number of leaf fragments for each experiment improved the reliability and reproducibility of the leaf disk assay. This cutter was also successfully applied to rice leaf disk assay, indicating the applicability to other plant spices.