Content of Two Major Steroidal Glycoalkaloids in Tomato (Solanum lycopersicum cv. Micro-Tom) Mutant Lines at Different Ripening Stages.

Esculeoside A and tomatine are two major steroidal alkaloids in tomato fruit (Solanum lycopersicum) that exhibit anti-inflammatory, anticancer, and anti-hyperlipidemia activities. Tomatine contained in immature tomato fruit is converted to esculeoside A as the fruit matures. To develop new tomato varieties based on the content analysis of functional secondary metabolites, 184 mutant lines were generated from the original cultivar (S. lycopersicum cv. Micro-Tom) by radiation breeding. Ultra-performance liquid chromatography coupled with evaporative light scattering detector was used to identify the mutant lines with good traits by analyzing tomatine and esculeoside A content. Compared with the original cultivar, candidates for highly functional cultivars with high esculeoside A content were identified in the mature fruit of the mutant lines. The mutant lines with low and high tomatine content at an immature stage were selected as edible cultivars due to toxicity reduction and as a source of tomatine with various pharmacological activities, respectively. During the process of ripening from green to red tomatoes, the rate of conversion of tomatine to esculeoside A was high in the green tomatoes with a low tomatine content, whereas green tomatoes with a high tomatine content exhibited a low conversion rate. Using methanol extracts prepared from unripe and ripe fruits of the original cultivar and its mutant lines and two major compounds, we examined their cytotoxicity against FaDu human hypopharynx squamous carcinoma cells. Only tomatine exhibited cytotoxicity with an IC50 value of 5.589 µM, whereas the other samples did not exhibit cytotoxicity. Therefore, radiation breeding represents a useful tool for developing new cultivars with high quality, and metabolite analysis is applicable for the rapid and objective selection of potential mutant lines.

Identification of Loci Governing Agronomic Traits and Mutation Hotspots via a GBS-Based Genome-Wide Association Study in a Soybean Mutant Diversity Pool.

In this study, we performed a genotyping-by-sequencing analysis and a genome-wide association study of a soybean mutant diversity pool previously constructed by gamma irradiation. A GWAS was conducted to detect significant associations between 37,249 SNPs, 11 agronomic traits, and 6 phytochemical traits. In the merged data set, 66 SNPs on 13 chromosomes were highly associated (FDR p < 0.05) with the following 4 agronomic traits: days of flowering (33 SNPs), flower color (16 SNPs), node number (6 SNPs), and seed coat color (11 SNPs). These results are consistent with the findings of earlier studies on other genetic features (e.g., natural accessions and recombinant inbred lines). Therefore, our observations suggest that the genomic changes in the mutants generated by gamma irradiation occurred at the same loci as the mutations in the natural soybean population. These findings are indicative of the existence of mutation hotspots, or the acceleration of genome evolution in response to high doses of radiation. Moreover, this study demonstrated that the integration of GBS and GWAS to investigate a mutant population derived from gamma irradiation is suitable for dissecting the molecular basis of complex traits in soybeans.

The Unstable Restorer-of-fertility locus in pepper (Capsicum annuum. L) is delimited to a genomic region containing PPR genes.

KEY MESSAGE: Unstable Restorer-of-fertility (Rfu), conferring unstable fertility restoration in the pepper CGMS system, was delimited to a genomic region near Rf and is syntenic to the PPR-like gene-rich region in tomato. The use of cytoplasmic-genic male sterility (CGMS) systems greatly increases the efficiency of hybrid seed production. Although marker development and candidate gene isolation have been performed for the Restorer-of-fertility (Rf) gene in pepper (Capsicum annuum L.), the broad use of CGMS systems has been hampered by the instability of fertility restoration among pepper accessions, especially sweet peppers, due to the widespread presence of the Unstable Restorer-of-fertility (Rfu) locus. Therefore, to investigate the genetic factors controlling unstable fertility restoration in sweet peppers, we developed a segregation population (BC4F5) from crosses using a male-sterile line and an Rfu-containing line. Segregation did not significantly deviate from a 3:1 ratio for unstable fertility restoration to sterility, indicating single dominant locus control for unstable fertility restoration in this population. Genetic mapping delimited the Rfu locus to a 398 kb genomic region on chromosome 6, which is close to but different from the previously identified Rf-containing region. The Rfu-containing region harbors a pentatricopeptide repeat (PPR) gene, along with 10 other candidate genes. In addition, this region is syntenic to the genomic region containing the largest number of Rf-like PPR genes in tomato. Therefore, the dynamic evolution of PPR genes might be responsible for both the restoration and instability of fertility in pepper. During genetic mapping, we developed various molecular markers, including one that co-segregated with Rfu. These markers showed higher accuracy for genotyping than previously developed markers, pointing to their possible use in marker-assisted breeding of sweet peppers.

Time-Series Growth Prediction Model Based on U-Net and Machine Learning in Arabidopsis.

Yield prediction for crops is essential information for food security. A high-throughput phenotyping platform (HTPP) generates the data of the complete life cycle of a plant. However, the data are rarely used for yield prediction because of the lack of quality image analysis methods, yield data associated with HTPP, and the time-series analysis method for yield prediction. To overcome limitations, this study employed multiple deep learning (DL) networks to extract high-quality HTTP data, establish an association between HTTP data and the yield performance of crops, and select essential time intervals using machine learning (ML). The images of Arabidopsis were taken 12 times under environmentally controlled HTPP over 23 days after sowing (DAS). First, the features from images were extracted using DL network U-Net with SE-ResXt101 encoder and divided into early (15-21 DAS) and late (∼21-23 DAS) pre-flowering developmental stages using the physiological characteristics of the Arabidopsis plant. Second, the late pre-flowering stage at 23 DAS can be predicted using the ML algorithm XGBoost, based only on a portion of the early pre-flowering stage (17-21 DAS). This was confirmed using an additional biological experiment (P < 0.01). Finally, the projected area (PA) was estimated into fresh weight (FW), and the correlation coefficient between FW and predicted FW was calculated as 0.85. This was the first study that analyzed time-series data to predict the FW of related but different developmental stages and predict the PA. The results of this study were informative and enabled the understanding of the FW of Arabidopsis or yield of leafy plants and total biomass consumed in vertical farming. Moreover, this study highlighted the reduction of time-series data for examining interesting traits and future application of time-series analysis in various HTPPs.

Mutagenic Effect of Proton Beams Characterized by Phenotypic Analysis and Whole Genome Sequencing in Arabidopsis.

Protons may have contributed to the evolution of plants as a major component of cosmic-rays and also have been used for mutagenesis in plants. Although the mutagenic effect of protons has been well-characterized in animals, no comprehensive phenotypic and genomic analyses has been reported in plants. Here, we investigated the phenotypes and whole genome sequences of Arabidopsis M2 lines derived by irradiation with proton beams and gamma-rays, to determine unique characteristics of proton beams in mutagenesis. We found that mutation frequency was dependent on the irradiation doses of both proton beams and gamma-rays. On the basis of the relationship between survival and mutation rates, we hypothesized that there may be a mutation rate threshold for survived individuals after irradiation. There were no significant differences between the total mutation rates in groups derived using proton beam or gamma-ray irradiation at doses that had similar impacts on survival rate. However, proton beam irradiation resulted in a broader mutant phenotype spectrum than gamma-ray irradiation, and proton beams generated more DNA structural variations (SVs) than gamma-rays. The most frequent SV was inversion. Most of the inversion junctions contained sequences with microhomology and were associated with the deletion of only a few nucleotides, which implies that preferential use of microhomology in non-homologous end joining was likely to be responsible for the SVs. These results show that protons, as particles with low linear energy transfer (LET), have unique characteristics in mutagenesis that partially overlap with those of low-LET gamma-rays and high-LET heavy ions in different respects.

Suggested doses of proton ions and gamma-rays for mutation induction in 20 plant species.

PURPOSE: Proton ions are expected to be used as a discriminative radiation source to induce different kinds of mutations than those produced by γ-rays and carbon ions; however, there is little systemic information about radiosensitivity in plants. MATERIALS AND METHODS: We analyzed the LD30, LD50, and RD50 values in response to proton ions and γ-rays using 20 plant species. Plant seeds were irradiated, and growth responses were measured one month after planting, except for cymbidium, for which in vitro rhizomes were irradiated. The rhizomes were analyzed at six and nine months after subculturing. RESULTS: Resistance to proton ions and γ-rays was observed in Chinese cabbage, watermelon, and melon, while Japanese atractylodes, naked barley, and lentil were susceptible. Plants belonging to the Brassicaceae and Cucurbitaceae families were highly resistant to radiation, and plants belonging to the Compositae and Poaceae families were highly susceptible. In addition, plants with genome sizes greater than 8,000 Mbp were highly sensitive to radiation, but there was no clear relationship between radiosensitivity and genome size in plants with genomes smaller than 2,500 Mbp. CONCLUSIONS: The biological effectiveness of proton ions was greater than that of γ-rays in 16 plant species, indicating that they could be used as a discriminative radiation source to induce mutations compared with γ-rays.

Measurement of Genetic Mobility Using a Transposon-Based Marker System in Sorghum.

Transposable elements (TEs) are ubiquitous repetitive components of eukaryotic organisms that show mobility in the genome against diverse stresses. TEs contribute considerably to the size, structure, and plasticity of genomes and also play an active role in genome evolution by helping their hosts adapt to novel conditions by conferring useful characteristics. We developed a simple and rapid method for investigation of genetic mobility and diversity among TEs in combination with a target region amplification polymorphism (TE-TRAP) marker system in gamma-irradiated sorghum mutants. The TE-TRAP marker system reveals a high level of genetic diversity, which provides a useful marker resource for genetic mobility research.

Effects of Acute and Chronic Gamma Irradiation on the Cell Biology and Physiology of Rice Plants.

The response to gamma irradiation varies among plant species and is affected by the total irradiation dose and dose rate. In this study, we examined the immediate and ensuing responses to acute and chronic gamma irradiation in rice (Oryza sativa L.). Rice plants at the tillering stage were exposed to gamma rays for 8 h (acute irradiation) or 10 days (chronic irradiation), with a total irradiation dose of 100, 200, or 300 Gy. Plants exposed to gamma irradiation were then analyzed for DNA damage, oxidative stress indicators including free radical content and lipid peroxidation, radical scavenging, and antioxidant activity. The results showed that all stress indices increased immediately after exposure to both acute and chronic irradiation in a dose-dependent manner, and acute irradiation had a greater effect on plants than chronic irradiation. The photosynthetic efficiency and growth of plants measured at 10, 20, and 30 days post-irradiation decreased in irradiated plants, i.e., these two parameters were more severely affected by acute irradiation than by chronic irradiation. In contrast, acutely irradiated plants produced seeds with dramatically decreased fertility rate, and chronically irradiated plants failed to produce fertile seeds, i.e., reproduction was more severely affected by chronic irradiation than by acute irradiation. Overall, our findings suggest that acute gamma irradiation causes instantaneous and greater damage to plant physiology, whereas chronic gamma irradiation causes long-term damage, leading to reproductive failure.

Genotype-by-Sequencing Analysis of Mutations and Recombination in Pepper Progeny of Gamma-Irradiated Gametophytes.

The irradiation of dry seeds is the most widely-used irradiation method for improving seed-propagated crops; however, the irradiation of other tissues also has useful effects. The irradiation of plant reproductive organs, rather than seeds, for mutation breeding has advantages, such as producing non-chimeric progeny. However, the mutation frequency and spectrum produced using this method have not been analyzed on a genome-wide level. We performed a genotype-by-sequencing analysis to determine the frequencies of single-base substitutions and small (1-2 bp) insertions and deletions in hot pepper (Capsicum annuum L.) plants derived from crosses using gamma-irradiated female or male gametophytes. The progeny of irradiated gametophytes showed similar or higher DNA mutation frequencies, which were dependent on the irradiation dose and irradiated tissue, and less biased single base substitutions than progeny of irradiated seeds. These characteristics were expected to be beneficial for development of mutation population with a high frequency of small DNA mutations and performing reverse-genetics-based mutation screening. We also examined the possible use of this irradiation method in manipulating the meiotic recombination frequency; however, no statistically significant increase was detected. Our results provide useful information for further research and breeding using irradiated gametophytes.

Development of a Rapid Selection System for Salt-Resistant Mutants of Nicotiana benthamiana through Protoplast Culture after Gamma Irradiation.

We aimed to develop a novel technology capable of rapidly selecting mutant plant cell lines. Salt resistance was chosen as a rapid selection trait that is easily applicable to protoplast-derived cell colonies. Mesophyll protoplasts were cultured in a medium supplemented with 0, 50, 100, 150, 200, 250, and 300 mM NaCl. At NaCl concentrations ≥ 100 mM, cell colony formation was strongly inhibited after 4 weeks of culture. Tobacco protoplasts irradiated with 0, 50, 100, 200, and 400 Gy were then cultured to investigate the effects of radiation intensity on cell division. The optimal radiation intensity was 50 Gy. To develop salt-resistant tobacco mutant plants, protoplasts irradiated with 50 Gy were cultured in a medium containing 100 mM NaCl. The efficiency of cell colony formation from these protoplasts was approximately 0.002%. A salt-resistant mutant callus was selected and proliferated in the same medium and then transferred to a shoot inducing medium for adventitious shoot formation. The obtained shoots were then cultured in a medium supplemented with 200 mM NaCl and developed into normal plantlets. This rapid selection technology for generating salt-resistant tobacco mutants will be useful for the development of crop varieties resistant to environmental stresses.

Comparative Analysis of Volatile Compounds of Gamma-Irradiated Mutants of Rose (Rosa hybrida).

Roses are one of the most important floricultural crops, and their essential oils have long been used for cosmetics and aromatherapy. We investigated the volatile compound compositions of 12 flower-color mutant variants and their original cultivars. Twelve rose mutant genotypes were developed by treatment with 70 Gy of 60Co gamma irradiation of six commercial rose cultivars. Essential oils from the flowers of the 18 genotypes were analyzed by gas chromatography-mass spectrometry. Seventy-seven volatile compounds were detected, which were categorized into six classes: Aliphatic hydrocarbons, aliphatic alcohols, aliphatic ester, aromatic compounds, terpene alcohols, and others. Aliphatic (hydrocarbons, alcohols, and esters) compounds were abundant categories in all rose flowers. The CR-S2 mutant had the highest terpene alcohols and oil content. Three (CR-S1, CR-S3, and CR-S4) mutant genotypes showed higher ester contents than their original cultivar. Nonacosane, 2-methylhexacosane, and 2-methyltricosane were major volatile compounds among all genotypes. Hierarchical cluster analysis (HCA) of the rose genotypes gave four groups according to grouping among the 77 volatile compounds. In addition, the principal component analysis (PCA) model was successfully applied to distinguish most attractive rose lines. These findings will be useful for the selection of rose genotypes with improved volatile compounds.

Dramatic Increase in Content of Diverse Flavonoids Accompanied with Down-Regulation of F-Box Genes in a Chrysanthemum (Chrysanthemum × morifolium (Ramat.) Hemsl.) Mutant Cultivar Producing Dark-Purple Ray Florets.

Anthocyanins (a subclass of flavonoids) and flavonoids are crucial determinants of flower color and substances of pharmacological efficacy, respectively, in chrysanthemum. However, metabolic and transcriptomic profiling regarding flavonoid accumulation has not been performed simultaneously, thus the understanding of mechanisms gained has been limited. We performed HPLC-DAD-ESI-MS (high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization mass spectrometry) and transcriptome analyses using "ARTI-Dark Chocolate" (AD), which is a chrysanthemum mutant cultivar producing dark-purple ray florets, and the parental cultivar "Noble Wine" for metabolic characterization and elucidation of the genetic mechanism determining flavonoid content. Among 26 phenolic compounds identified, three cyanidins and eight other flavonoids were detected only in AD. The total amounts of diverse flavonoids were 8.0 to 10.3 times higher in AD. Transcriptome analysis showed that genes in the flavonoid biosynthetic pathway were not up-regulated in AD at the early flower stage, implying that the transcriptional regulation of the pathway did not cause flavonoid accumulation. However, genes encoding post-translational regulation-related proteins, especially F-box genes in the mutated gene, were enriched among down-regulated genes in AD. From the combination of metabolic and transcriptomic data, we suggest that the suppression of post-translational regulation is a possible mechanism for flavonoid accumulation in AD. These results will contribute to research on the regulation and manipulation of flavonoid biosynthesis in chrysanthemum.

Epigenetic Variation Induced by Gamma Rays, DNA Methyltransferase Inhibitors, and Their Combination in Rice.

DNA methylation plays important roles in the regulation of gene expression and maintenance of genome stability in many organisms, including plants. In this study, we treated rice with gamma rays (GRs) and DNA methyltransferase inhibitors (DNMTis) to induce variations in DNA methylation and evaluated epigenetic diversity using methylation-sensitive amplified polymorphism (MSAP) and transposon methylation display (TMD) marker systems. Comparative and integrated analyses of the data revealed that both GRs and DNMTis alone have epimutagenic effects and that combined treatment enhanced these effects. Calculation of methylation rates based on band scoring suggested that both GRs and DNMTis induce epigenetic diversity by demethylation in a dose-dependent manner, and combined treatment can induce variations more synergistically. The difference in the changes in full and hemi-methylation rates between MSAP and TMD is presumed to be caused by the different genomic contexts of the loci amplified in the two marker systems. Principal coordinate, phylogenic, and population structure analyses commonly yielded two clusters of individuals divided by DNMTi treatment. The clustering pattern was more apparent in TMD, indicating that DNMTis have a stronger effect on hypermethylated repetitive regions. These findings provide a foundation for understanding epigenetic variations induced by GRs and DNMTis and for epigenetic mutation breeding.

High-Throughput Phenotyping (HTP) Data Reveal Dosage Effect at Growth Stages in Arabidopsis thaliana Irradiated by Gamma Rays.

The effects of radiation dosages on plant species are quantitatively presented as the lethal dose or the dose required for growth reduction in mutation breeding. However, lethal dose and growth reduction fail to provide dynamic growth behavior information such as growth rate after irradiation. Irradiated seeds of Arabidopsis were grown in an environmentally controlled high-throughput phenotyping (HTP) platform to capture growth images that were analyzed with machine learning algorithms. Analysis of digital phenotyping data revealed unique growth patterns following treatments below LD50 value at 641 Gy. Plants treated with 100-Gy gamma irradiation showed almost identical growth pattern compared with wild type; the hormesis effect was observed >21 days after sowing. In 200 Gy-treated plants, a uniform growth pattern but smaller rosette areas than the wild type were seen (p < 0.05). The shift between vegetative and reproductive stages was not retarded by irradiation at 200 and 300 Gy although growth inhibition was detected under the same irradiation dose. Results were validated using 200 and 300 Gy doses with HTP in a separate study. To our knowledge, this is the first study to apply a HTP platform to measure and analyze the dosage effect of radiation in plants. The method enabled an in-depth analysis of growth patterns, which could not be detected previously due to a lack of time-series data. This information will improve our knowledge about the effects of radiation in model plant species and crops.

Development and Characterization of an Ethyl Methane Sulfonate (EMS) Induced Mutant Population in Capsicum annuum L.

Plant breeding explores genetic diversity in useful traits to develop new, high-yielding, and improved cultivars. Ethyl methane sulfonate (EMS) is a chemical widely used to induce mutations at loci that regulate economically essential traits. Additionally, it can knock out genes, facilitating efforts to elucidate gene functions through the analysis of mutant phenotypes. Here, we developed a mutant population using the small and pungent ornamental Capsicum annuum pepper "Micro-Pep". This accession is particularly suitable for mutation studies and molecular research due to its compact growth habit and small size. We treated 9500 seeds with 1.3% EMS and harvested 3996 M2 lines. We then selected 1300 (32.5%) independent M2 families and evaluated their phenotypes over four years. The mutants displayed phenotypic variations in plant growth, habit, leaf color and shape, and flower and fruit morphology. An experiment to optimize Targeting Induced Local Lesions IN Genomes (TILLING) in pepper detected nine EMS-induced mutations in the eIF4E gene. The M2 families developed here exhibited broad phenotypic variation and should be valuable genetic resources for functional gene analysis in pepper molecular breeding programs using reverse genetics tools, including TILLING.

Transcriptome analysis to identify candidate genes associated with the yellow-leaf phenotype of a Cymbidium mutant generated by γ-irradiation.

Leaf color is an important agronomic trait in flowering plants, including orchids. However, factors underlying leaf phenotypes in plants remain largely unclear. A mutant displaying yellow leaves was obtained by the γ-ray-based mutagenesis of a Cymbidium orchid and characterized using RNA sequencing. A total of 144,918 unigenes obtained from over 25 million reads were assigned to 22 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes database. In addition, gene ontology was used to classify the predicted functions of transcripts into 73 functional groups. The RNA sequencing analysis identified 2,267 differentially expressed genes between wild-type and mutant Cymbidium sp. Genes involved in the chlorophyll biosynthesis and degradation, as well as ion transport, were identified and assayed for their expression levels in wild-type and mutant plants using quantitative real-time profiling. No critical expression changes were detected in genes involved in chlorophyll biosynthesis. In contrast, seven genes involved in ion transport, including two metal ion transporters, were down-regulated, and chlorophyllase 2, associated with chlorophyll degradation, was up-regulated. Together, these results suggest that alterations in chlorophyll metabolism and/or ion transport might contribute to leaf color in Cymbidium orchids.

Effects of the total dose and duration of γ-irradiation on the growth responses and induced SNPs of a Cymbidium hybrid.

Purpose: Ionizing radiation has been used for developing new cultivars of diverse plant species, including Cymbidium orchid species. The effects of the total dose on mutation induction have been investigated; however, there is relatively little research on the influence of the dose rate or irradiation duration.Materials and methods: Thus, we analyzed the effects of the total dose and irradiation duration on the growth of Cymbidium hybrid RB001 protocorm-like bodies (PLBs). We completed a genotyping-by-sequencing analysis to compare the induced SNPs among five γ-irradiated populations with similar growth responses (LD50) to γ-rays.Results: The optimal time to assess the effects of the γ-irradiation was at 6 months after the treatment. On the basis of the survival rate of γ-irradiated PLBs, the optimal doses (LD50) for each irradiation duration were estimated: 1 h, 16.1 Gy; 4 h, 23.6 Gy; 8 h, 37.9 Gy; 16 h, 37.9 Gy; and 24 h, 40.0 Gy. The estimated optimal doses were duration-dependent at irradiation durations shorter than 8 h, but not at irradiation durations exceeding 8 h. A SNP comparison revealed a lack of significant differences among the mutations induced by γ-irradiations.Conclusions: These results indicate the irradiation duration affects PLB growth in response to γ-rays. Moreover, the mutations induced by a short-term treatment may be similar to those induced by a treatment over a longer period.

Mitotypes Based on Structural Variation of Mitochondrial Genomes Imply Relationships With Morphological Phenotypes and Cytoplasmic Male Sterility in Peppers.

Plant mitochondrial genomes characteristically contain extensive structural variation that can be used to define and classify cytoplasm types. We developed markers based on structural variation in the mitochondrial genomes of fertile and cytoplasmic male sterility (CMS) pepper lines and applied them to a panel of Capsicum accessions. We designed a total of 20 sequence characterized amplified region (SCAR) markers based on DNA rearrangement junctions or cytoplasm-specific segments that did not show high similarity to any nuclear mitochondrial DNA segments. We used those markers to classify the mitotypes of 96 C. annuum accessions into 15 groups. Precise genotyping of other Capsicum species (C. frutescens, C. chinense, and C. baccatum) was hampered because of various stoichiometric levels of marker amplicons. We developed a multiplex PCR system based on four of the markers that efficiently classified the C. annuum accessions into five mitotype groups. Close relationships between specific mitotypes and morphological phenotypes implied that diversification or domestication of C. annuum germplasm might have been accompanied by structural rearrangements of mitochondrial DNA or the selection of germplasms with specific mitotypes. Meanwhile, CMS lines shared the same amplification profile of markers with another mitotype. Further analysis using mitochondrial DNA (mtDNA) markers based on single-nucleotide polymorphisms (SNPs) or insertions and deletions (InDels) and CMS-specific open reading frames (orfs) provided new information about the origin of the CMS-specific mitotype and evaluation of candidates for CMS-associated genes, respectively.

Comparative Analysis of Phytochemical Composition of Gamma-Irradiated Mutant Cultivars of Chrysanthemum morifolium.

The flowers of chrysanthemum species are used as a herbal tea and in traditional medicine. In addition, members of the genus have been selected to develop horticultural cultivars of diverse floral colors and capitulum forms. In this research, we investigated the phytochemical composition of eight gamma-irradiation mutant cultivars of Chrysanthemum morifolium and their original cultivars. The mutant chrysanthemum cultivars were generated by treatment with various doses of 60Co gamma irradiation of stem cuttings of three commercial chrysanthemum cultivars as follows: 'ARTI-Dark Chocolate' (50Gy), 'ARTI-Purple Lady' (30 Gy), and 'ARTI-Yellow Star' (50 Gy) derived from 'Noble Wine'; 'ARTI-Red Star' (50 Gy) and 'ARTI-Rising Sun' (30 Gy) from 'Pinky'; 'ARTI-Purple' (40 Gy) and 'ARTI-Queen' (30 Gy) from 'Argus'; and 'ARTI-Rollypop' (70 Gy) from 'Plaisir d'amour'. Quantitative analysis of flavonoids, phenolic acids, anthocyanins, and carotenoids in the flowers of the 12 chrysanthemum cultivars was performed using high performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry (HPLC-DAD-ESIMS). Essential oils from the flowers of these cultivars were analyzed by gas chromatography-mass spectrometry (GC-MS). The mutant cultivars, 'ARTI-Dark Chocolate', 'ARTI-Purple Lady', 'ARTI-Purple', and 'ARTI-Queen' showed higher total amounts of flavonoid and phenolic acid compared with those of the respective original cultivars. The mutant cultivars, 'ARTI-Dark Chocolate', 'ARTI-Purple Lady' and 'ARTI-Purple', which produce purple to pink petals, contained more than two-times higher amounts of anthocyanins compared with those of their original cultivars. Of the mutant cultivars, 'ARTI-Yellow Star' in which petal color was changed to yellow, showed the greatest accumulation of carotenoids. Ninety-nine volatile compounds were detected, of which hydrocarbons and terpenoids were abundant in all cultivars analyzed. This is the first report that demonstrated the phytochemical analysis of novel chrysanthemum cultivars derived from C. morifolium hydrid using HPLC-DAD-ESIMS and GC-MS. These findings suggest that the selected mutant chrysanthemum cultivars show potential as a functional source of phytochemicals associated with the abundance of health-beneficial components, as well as good source for horticulture and pigment industries.

Isolation of soluble epoxide hydrolase inhibitor of capsaicin analogs from Capsicum chinense Jacq. cv. Habanero.

Capsaicin (1) and dihydrocapsaicin (2) were isolated from the aerial parts of Capsicum chinense Jacq. cv. Habanero. In in vitro studies, the exhibited potent inhibitory activity against soluble epoxide hydrolase (sEH), with IC50 values of 5.6 ±â€¯1.2 and 7.3 ±â€¯0.7 µM. Enzyme kinetics suggested that the two compounds (1 and 2) were competitive inhibitors with Ki values of 2.5 ±â€¯1.0 and 4.7 ±â€¯2.3 µM, respectively. Molecular docking and molecular dynamic studies showed that capsaicin analogs (1 and 2) bound strongly with Asp335, Tyr383, and Tyr466 residues in the active site. These results suggest that the two inhibitors (1 and 2) represent potentially therapeutic inhibitors of sEH.