DNA methylation-based organ tissue identification: Marker identification, SNaPshot multiplex assay development, and interlaboratory comparison.

Identifying body fluids and organ tissues is highly significant as they can offer crucial evidence in criminal investigations and aid the court in making informed decisions, primarily through evaluating the biological source and possibly at the activity level up to death or fatal damage. In this study, organ tissue-specific CpG markers were identified from Illumina's methylation EPIC array data of nine organ tissues, including epidermis, dermis, heart, skeletal muscle, blood, kidney, brain, lung, and liver, from autopsies of 10 Koreans. Through the validation test using 43 samples, 18 hypomethylation markers, with two markers for each organ tissue type, were selected to construct a SNaPshot assay. Two multiplex assays involving forward and reverse SBE primers were designed to help investigators accurately determine the organ origin of the analyzed tissue samples through repeated analysis of the same PCR products for markers. The developed multiplex demonstrated high accuracy, achieving 100.0 % correct detection of the presence of nine organ tissue types in 88 samples from autopsies of 10 Asians. However, two lung samples showed additional positive indications of the presence of blood. An interlaboratory comparison using 80 autopsy samples (heart, skeletal muscle, blood, kidney cortex, kidney medulla, brain, lung, and liver) from 10 individuals in Germany revealed overall comparable results with correct detection of the presence of eight organ tissue types in 92.5 % samples (74 of 80 samples). In the case of six samples, it was impossible to determine the correct tissue successfully due to drop-outs of unmethylation signals at target tissue marker loci. One of these lung samples revealed only non-intended off-target signals for blood. The observed differences might be due to differences in sample collection during routine autopsy, technical differences due to the PCR cycler, and the threshold used for signal calling. Indicating the presence of additional tissue type and off-target unmethylation signals seems alleviated by applying more stringent hypomethylation thresholds. Therefore, the developed SNaPshot multiplex assays will be valuable for forensic investigators dealing with organ tissue identification, as well as for prosecutors and defense aiming to establish the circumstances that occurred at the crime scene.

Metabolic Profiling in Plasma and Brain Induced by 17ß-Estradiol Supplementation in Ovariectomized Mice.

17ß-Estradiol is an ovarian hormone that regulates energy circulation and storage by acting on the central nervous system. However, the metabolic differences between the blood and brain when stimulated by 17ß-estradiol are poorly understood. Moreover, research using menopause-induced models to investigate primary metabolites in the blood and brain is limited. Thus, this study aimed to identify metabolic changes in the plasma and brain resulting from 17ß-estradiol supplementation in an estrogen-deficient mouse model. Three groups of mice were utilized: sham-operated mice (Sham), ovariectomized mice (OVX), and ovariectomized mice that received a weekly supplementation of 17ß-estradiol (E2). Plasma and brain samples from these mice were subjected to metabolic analysis using gas chromatography-time-of-flight-mass spectrometry. Compared with the plasma samples from the Sham and OVX groups, the plasma samples from the E2 group contained higher contents of branched-chain amino acids (BCAAs), such as valine, isoleucine, and leucine. Meanwhile, the brain samples from the E2 group contained higher contents of most metabolites, including BCAAs, neurotransmitters, tricarboxylic acid cycle intermediates, and fatty acids, than those from the two other groups. This study is the first to reveal differences in energy metabolism induced by 17ß-estradiol supplementation through brain metabolic profiling of ovariectomized mice, emphasizing the importance of brain metabolic profiling in menopausal hormone research.

Age prediction using DNA methylation of Y-chromosomal CpGs in semen samples.

In cases of sexual assault, the evidence often exists as a mixture of female and male body fluids, and in many cases, contains a higher proportion of female body fluids than males. In these cases, Y-STR, rather than autosomal STRs, can provide useful information. It becomes very difficult to identify the true suspect if there is no match among known suspects or if a match exists for two or more suspects, e.g. two suspects from the same paternal lineage. However, age prediction using the DNA methylation of Y-chromosomal CpGs can help narrow the search for unknown suspects and discriminate between older and younger suspects. Therefore, the DNA methylation profiles of semen samples from 56 healthy Korean males were generated using Illumina's Infinium MethylationEPIC BeadChip Array. Among the ten identified age-associated CpG markers located in the Y-chromosome, nine were used to construct age prediction models. The identified markers were further investigated in the MPS analysis of 147 semen samples, and the multiplex assay was validated with the reliability, reproducibility and sensitivity tests. Several age prediction models were constructed using the MPS data with the multiple linear regression, stepwise linear regression, ridge linear regression, lasso regression, elastic net linear regression and support vector machine analyses, and all showed MAEs of 5 to 7 years in the test set samples. Six single-source female samples were also subjected to MPS analysis but showed very low coverage that could not affect the analysis of the mixed samples. Therefore, the age prediction models of the present study are expected to provide useful investigative leads, especially in mixed male and female samples from sexual assault cases.

Application of array-based age prediction models to post-mortem tissue samples.

Since DNA methylation at specific CpG sites exhibits a strong age association, researchers have developed numerous age prediction models based on the methylation BeadChip array. These models harness epigenetic clocks that hold the potential to narrow down the search range for unknown suspects and unidentified victims. This study collected 180 post-mortem tissue samples comprising nine tissue types (blood, brain, heart, lung, liver, kidney, muscle, epidermis, and dermis) from autopsies of 20 Koreans aged 18-78. Subsequently, DNA methylation profiling was conducted using the Infinium MethylationEPIC array. We tested several array-based age prediction models using the data obtained from various tissues. The pan-tissue clock exhibited a moderately accurate prediction across all nine tissue types (MAE = 8.7 years, r = 0.88). Notably, the DNAm ages of the Hannum clock, the skin & blood clock, and the Zhang clock strongly correlated with the actual age in blood samples (MAE < approximately 5 years, r > 0.9). PhenoAge yielded an MAE of 10.1 years and an r-value of 0.92. The muscle-specific epigenetic clock, the MEAT package, demonstrated high prediction accuracy in muscle samples (MAE = 4.7 years, r = 0.93). Those previously reported array-based age prediction models were mainly constructed in Europeans but performed well in Koreans. In addition, tests involving various quantities of DNA and fragmented DNA have shown that DNA quantity and quality affected methylation measurements and age prediction results. However, robust age prediction models exist under low amounts of DNA and fragmented DNA conditions.

Production of Phenolic Compounds and Antioxidant Activity in Hairy Root Cultures of Salvia plebeia.

Salvia plebeia (Lamiaceae) is a medicinal plant containing diverse bioactive constituents that have biological properties. In this study, we determined the optimal conditions (media and auxin) for the hairy root culture of S. plebeia for the growth and accumulation of phenolic compounds and evaluated its antioxidant activities. Rosmarinic acid and five phenylpropanoids were detected using high-performance liquid chromatography. The hairy roots grown in 1/2 SH medium with 1 mg/L NAA had a high level of rosmarinic acid content. Hairy roots cultured in 1 mg/L NAA had the highest total content of five phenylpropanoids. Compared to wild-type roots grown in the field, hairy roots (NAA 1) expressed similar levels of rosmarinic acid but significantly enhanced phenylpropanoid accumulation. Furthermore, the total phenolic content and total flavonoid content of hairy roots (NAA 1) were 2.22 and 1.73 times higher than those of wild-type roots. In the results of DPPH, ABTS, and reducing power assays, the hairy roots (NAA 1) showed higher free radical scavenging effects and reduction potential than the wild-type roots. These results suggest that S. plebeia hairy roots cultured under optimal conditions, which exhibit enhanced phenolic compound accumulation and antioxidant activity, can potentially be used as sources of antioxidants.

Metabolite Changes in Soybean (Glycine max) Leaves during the Entire Growth Period.

Although soybean (Glycine max) leaves generate building blocks to produce seeds, a comprehensive understanding of the metabolic changes in soybean leaves during the entire growth stages is limited. Here, we investigated the metabolite changes in soybean leaves from five cultivars among four vegetative (V) and eight reproductive (R) stages using metabolite profiling coupled with chemometrics. Principal component analysis (PCA) of all samples showed a clear separation by growth stage. The total amount of monosaccharides and organic acids for energy production were highly detected in the V stage samples, accumulating in concentrations 2.5 and 1.7 times higher than in the R stage samples, respectively. The results of partial least-squares-discriminant analysis (PLS-DA) revealed a clear separation from R1 to R5 by the first PLS, suggesting significant alterations in the metabolic networks up to R5. After flowering, the stage of seed formation, R5, was associated with lower levels of most amino acids and an accumulation of phytosterols. The negative correlation observed between amino acids and phytosterol levels suggests a sophisticated coordination between carbon and nitrogen metabolism in plant, ensuring and supporting optimal growth (r = -0.50085, P = 0.0001). In addition, R-stage samples had decreased monosaccharide levels, indicating redistribution to seeds and senescence-related metabolite changes. Thus, metabolite profiling coupled with chemometrics could be a useful tool for investigating alterations in metabolic networks during various plant growth and development stages. Furthermore, we observed variations in flavonoid contents among the different cultivars. The results could be a basis of further studies on the source-sink interactions in the plant system.

Metabolite profiles and biological activities of different phenotypes of Chinese cabbage (Brassica rapa ssp. Pekinensis).

Chinese cabbage is considered as one of the most important cruciferous vegetables in South Korea because of its use in salads, kimchi, and Korean cuisine. Secondary metabolites were quantified in three Chinese cabbage varieties: 65065, interspecific hybrid of Chinese cabbage × red cabbage exhibiting a deep purple color; 85772, interspecific hybrid of Chinese cabbage × red mustard exhibiting a reddish-purple color; and a typical Chinese green cabbage cultivar "CR Carotene" (Brassica rapa subsp. pekinensis cv. CR Carotene). A total of 54 metabolites (2 amines, 2 sugar alcohols, 2 sugar phosphates, 6 carbohydrates, 18 amino acids, 13 organic acids, 8 phenolic compounds, and 3 carotenoids) were detected in 85772. Of them, 52 metabolites excluding ß-carotene and 9-cis-ß-carotene, and 51 metabolites excluding leucine, ß-carotene, and 9-cis-ß-carotene, were detected in 65065 and CR Carotene, respectively. Amino acid content was the highest in 85772, followed by 65065 and CR Carotene. The cultivars 65065 and 85772 contained high levels of phenolic compounds and total anthocyanins. Cyanidin-, pelargonidin-, and petunidin-type anthocyanins were detected in 65065 and 85772. However, delphinidin-type anthocyanins which typically impart a deep purple color were identified only in the deep purple phenotype 65065. Furthermore, the total anthocyanin content was the highest in 85772 (4.38 ± 0.65 mg g -1 dry weight) followed by that in 65065 (3.72 ± 0.52 mg g-1 dry weight). Antibacterial and antioxidant analyses revealed remarkable antibacterial effects of the purple cultivars against pathogens Vibrio parahaemolyticus (KCTC 2471), Bacillus cereus (KCTC 3624), Pseudomonas aeruginosa (KCCM 11803), Staphylococcus aureus (KCTC 3881), Chryseobacterium gleum (KCTC 2094), and Proteus mirabilis (KCTC 2510)] and methicillin-resistant pathogenic strains of Pseudomonas aeruginosa (0826, 0225, 0254, 1113, 1378, 1731, p01827, and p01828) compared with the antibacterial effects of CR Carotene. Furthermore, 65065 and 85772 exhibited significantly higher antioxidant activity than that of the CR Carotene. Therefore, the novel purple Chinese cabbages (65065 and 85772), derived from interspecific hybridization, are potentially favorable alternatives to the typical green Chinese cabbage, given the higher content of amino acids, phenolic compounds, anthocyanins, and carotenoids, as well as an increased ability to scavenge free radicals and inhibit pathogen growth.

LED Lights Influenced Phytochemical Contents and Biological Activities in Kale (Brassica oleracea L. var. acephala) Microgreens.

Light-emitting diodes (LEDs) are regarded as an effective artificial light source for producing sprouts, microgreens, and baby leaves. Thus, this study aimed to investigate the influence of different LED lights (white, red, and blue) on the biosynthesis of secondary metabolites (glucosinolates, carotenoids, and phenolics) and the biological effects on kale microgreens. Microgreens irradiated with white LEDs showed higher levels of carotenoids, including lutein, 13-cis-ß-carotene, α-carotene, ß-carotene, and 9-cis-ß-carotene, than those irradiated with red or blue LEDs. These findings were consistent with higher expression levels of carotenoid biosynthetic genes (BoPDS and BoZDS) in white-irradiated kale microgreens. Similarly, microgreens irradiated with white and blue LEDs showed slightly higher levels of glucosinolates, including glucoiberin, progoitrin, sinigrin, and glucobrassicanapin, than those irradiated with red LEDs. These results agree with the high expression levels of BoMYB28-2, BoMYB28-3, and BoMYB29 in white- and blue-irradiated kale microgreens. In contrast, kale microgreens irradiated with blue LEDs contained higher levels of phenolic compounds (gallic acid, catechin, ferulic acid, sinapic acid, and quercetin). According to the total phenolic content (TPC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition assays, the extracts of kale microgreens irradiated with blue LEDs had slightly higher antioxidant activities, and the DPPH inhibition percentage had a positive correlation with TPC in the microgreens. Furthermore, the extracts of kale microgreens irradiated with blue LEDs exhibited stronger antibacterial properties against normal pathogens and multidrug-resistant pathogens than those irradiated with white and red LEDs. These results indicate that white-LED lights are suitable for carotenoid production, whereas blue-LED lights are efficient in increasing the accumulation of phenolics and their biological activities in kale microgreens.

Retraction notice to "Centella asiatica extract in Carboxymethyl Cellulose at its optimal concentration improved wound healing in mice model" [Heliyon 8, (2022) Article e12031].

[This retracts the article DOI: 10.1016/j.heliyon.2022.e12031.].

WUSCHEL controls genotype-dependent shoot regeneration capacity in potato.

Plant cells can reprogram their fate. The combinatorial actions of auxin and cytokinin dedifferentiate somatic cells to regenerate organs, which can develop into individual plants. As transgenic plants can be generated from genetically modified somatic cells through these processes, cell fate transition is an unavoidable step in crop genetic engineering. However, regeneration capacity closely depends on the genotype, and the molecular events underlying these variances remain elusive. In the present study, we demonstrated that WUSCHEL (WUS)-a homeodomain transcription factor-determines regeneration capacity in different potato (Solanum tuberosum) genotypes. Comparative analysis of shoot regeneration efficiency and expression of genes related to cell fate transition revealed that WUS expression coincided with regeneration rate in different potato genotypes. Moreover, in a high-efficiency genotype, WUS silencing suppressed shoot regeneration. Meanwhile, in a low-efficiency genotype, regeneration could be enhanced through the supplementation of a different type of cytokinin that promoted WUS expression. Computational modeling of cytokinin receptor-ligand interactions suggested that the docking pose of cytokinins mediated by hydrogen bonding with the core residues may be pivotal for WUS expression and shoot regeneration in potatoes. Furthermore, our whole-genome sequencing analysis revealed core sequence variations in the WUS promoters that differentiate low- and high-efficiency genotypes. The present study revealed that cytokinin responses, particularly WUS expression, determine shoot regeneration efficiency in different potato genotypes.

Lipids and volatile organic compounds in sesame seeds and their relationships with environmental temperature-induced stress.

Sesame seeds contain several lipids and fragrances that offer health benefits. However, no studies have reported a relationship between the lipids or flavor compounds of sesame seeds and environmental factors. In this study, we aimed to identify this relationship by analyzing the contents of lipidic and flavor compounds in fifteen genotypes of sesame seeds grown in two cultivation regions (Jeonju and Miryang) and years (2018 and 2019). Herein, 17 lipids and 62 flavor compounds were detected. Multivariate statistical analyses revealed that the cultivation year had a larger influence on the contents of lipidic and flavor compounds than the cultivation region and genotype. Furthermore, heat stress due to high cultivation temperature in 2018 caused the accumulation of sugar and secondary metabolites, increased flavor-related substances, and inhibited the degradation of fatty acids. Our study is the first to demonstrate the metabolic changes in lipids and flavor components of sesame in response to environmental temperature changes affected by different cultivation years. Therefore, this study provides guidance for the cultivation of commercially advantageous sesame seeds in improving the quality of sesame seeds and their products.

Metabolic Discrimination between Adventitious Roots and Standard Medicinal Part of Atractylodes macrocephala Koidz. Using FT-IR Spectroscopy.

This study aims to examine the metabolic discrimination between in vitro grown adventitious roots and the standard medicinal parts of Atractylodes macrocephala. To achieve this goal, firstly, in vitro culture conditions of adventitious roots such as indole-3-butyric acid (IBA) concentrations, types of media, inorganic salt strength of culture medium, and elicitor types and concentrations were optimized. The optimal culture conditions for proliferation of adventitious roots was found to consist of Murashige and Skoog (MS) medium containing 5 mg L-1 IBA. Whole cell extracts from adventitious roots and the standard medicinal parts of A. macrocephala were subjected to Fourier transform infrared spectroscopy (FT-IR). Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) from FT-IR spectral data showed that adventitious roots and standard medicinal parts were clearly distinguished in the PCA and PLS-DA score plot. Furthermore, the overall metabolite pattern from adventitious roots was changed depending on the dose-dependent manner of chemicals. These results suggest that FT-IR spectroscopy can be applied as an alternative tool for the screening of higher metabolic root lines and for discriminating metabolic similarity between in vitro grown adventitious roots and the standard medicinal parts. In addition, the adventitious roots proliferation system established in this study can be directly applied as an alternative means for the commercial production of A. macrocephala.

The Effect of Light and Dark Treatment on the Production of Rosmarinic Acid and Biological Activities in Perilla frutescens Microgreens.

This study aimed to investigate the effect of light [a long-day photoperiod (16 h light/8 h dark cycle)] and dark treatment on the production of rosmarinic acid in P. frutescens microgreens and to determine its antioxidant and antibacterial activities. Microgreens of P. frutescens were grown under light and dark conditions and harvested after 10, 15, 20, and 25 days of each treatment. Although dry weight values of microgreens gradually increased from 10 to 25 days of both treatments, the microgreens grown under light treatment possessed slightly higher levels of dry weight than those grown in the dark. Rosmarinic acid and total phenolic content (TPC) were also analyzed using high-performance liquid chromatography (HPLC) and Folin-Ciocalteu assay. The accumulation patterns of rosmarinic acid and TPC gradually increased and decreased, respectively, in P. frutescens microgreens grown in continuous darkness. The highest accumulation was observed in microgreens grown for 20 days. However, rosmarinic acid and TPC values were not significantly different in microgreens grown under light conditions. According to the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical inhibition assay, the extracts of P. frutescens microgreens were confirmed to be strong antioxidants, and their ability to scavenge DPPH radicals was positively correlated with the total phenolic content in the microgreens after 10, 15, 20, and 25 days of both treatments. Considering the relatively higher values of dry weight, rosmarinic acid, TPC, and DPPH assay, P. frutescens microgreens after 20 days of darkness and 20 days of light treatment, respectively, were selected for screening antibacterial activity using nine pathogens. Both microgreen extracts showed strong antibacterial activity against pathogens. In particular, the extracts of microgreens grown for 20 days under light treatment showed higher antimicrobial effects. Therefore, the light treatments for 20 days, as well as the darkness treatment for 20 days, were the best conditions for P. frutescens microgreen production because of their high levels of dry weight, phenolics, and biological activities.

Impact of Light and Dark Treatment on Phenylpropanoid Pathway Genes, Primary and Secondary Metabolites in Agastache rugosa Transgenic Hairy Root Cultures by Overexpressing Arabidopsis Transcription Factor AtMYB12.

Agastache rugosa, otherwise called Korean mint, has a wide range of medicinal benefits. In addition, it is a rich source of several medicinally valuable compounds such as acacetin, tilianin, and some phenolic compounds. The present study aimed to investigate how the Tartary buckwheat transcription factor AtMYB12 increased the primary and secondary metabolites in Korean mint hairy roots cultured under light and dark conditions. A total of 50 metabolites were detected by using high-performance liquid chromatography (HPLC) and gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). The result showed that the AtMYB12 transcription factor upregulated the phenylpropanoid biosynthesis pathway genes, which leads to the highest accumulation of primary and secondary metabolites in the AtMYB12-overexpressing hairy root lines (transgenic) than that of the GUS-overexpressing hairy root line (control) when grown under the light and dark conditions. However, when the transgenic hairy root lines were grown under dark conditions, the phenolic and flavone content was not significantly different from that of the control hairy root lines. Similarly, the heat map and hierarchical clustering analysis (HCA) result showed that most of the metabolites were significantly abundant in the transgenic hairy root cultures grown under light conditions. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) showed that the identified metabolites were separated far based on the primary and secondary metabolite contents present in the control and transgenic hairy root lines grown under light and dark conditions. Metabolic pathway analysis of the detected metabolites showed 54 pathways were identified, among these 30 were found to be affected. From these results, the AtMYB12 transcription factor activity might be light-responsive in the transgenic hairy root cultures, triggering the activation of the primary and secondary metabolic pathways in Korean mint.

Alternative Plant Vitrification Solution A3-80% and Initial Ammonium-Free Regrowth Medium Enable Cryobanking of Chrysanthemum Germplasm.

Cryopreservation, storing biological material in liquid nitrogen (LN, -196 °C), offers a valuable option for the long-term conservation of non-orthodox seeds and vegetatively propagated species in the sector of agrobiodiversity and wild flora. Although large-scale cryobanking of germplasm collections has been increasing worldwide, the wide application of cryopreservation protocol is hampered by a lack of universal cryopreservation protocols, among others. This study established a systematic approach to developing a droplet-vitrification cryopreservation procedure for chrysanthemum shoot tips. The standard procedure includes two-step preculture with 10% sucrose for 31 h and with 17.5% sucrose for 16 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 40 min, cryoprotection with alternative plant vitrification solution A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose, w/v) at 0 °C for 60 min, and cooling and rewarming using aluminum foil strips. After unloading, a three-step regrowth procedure starting with an ammonium-free medium with 1 mg L-1 gibberellic acid (GA3) and 1 mg L-1 benzyl adenine (BA) followed by an ammonium-containing medium with and without growth regulators was essential for the development of normal plantlets from cryopreserved shoot tips. A pilot cryobanking of 154 accessions of chrysanthemum germplasm initiated with post-cryopreservation regeneration of 74.8%. This approach will facilitate the cryobanking of the largest Asteraceae family germplasm as a complementary long-term conservation method.

Metabolic Profiling of Primary and Secondary Metabolites in Kohlrabi (Brassica oleracea var. gongylodes) Sprouts Exposed to Different Light-Emitting Diodes.

Light-emitting diode (LED) technology is one of the most important light sources in the plant industry for enhancing growth and specific metabolites in plants. In this study, we analyzed the growth, primary and secondary metabolites of 10 days old kohlrabi (Brassica oleracea var. gongylodes) sprouts exposed to different LED light conditions. The results showed that the highest fresh weight was achieved under red LED light, whereas the highest shoot and root lengths were recorded below the blue LED light. Furthermore, high-performance liquid chromatography (HPLC) analysis revealed the presence of 13 phenylpropanoid compounds, 8 glucosinolates (GSLs), and 5 different carotenoids. The phenylpropanoid and GSL contents were highest under blue LED light. In contrast, the carotenoid content was found to be maximum beneath white LED light. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) of the 71 identified metabolites using HPLC and gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) showed a clear separation, indicating that different LEDs exhibited variation in the accumulation of primary and secondary metabolites. A heat map and hierarchical clustering analysis revealed that blue LED light accumulated the highest amount of primary and secondary metabolites. Overall, our results demonstrate that exposure of kohlrabi sprouts to blue LED light is the most suitable condition for the highest growth and is effective in increasing the phenylpropanoid and GSL content, whereas white light might be used to enhance carotenoid compounds in kohlrabi sprouts.

Metabolite Profiling to Evaluate Metabolic Changes in Genetically Modified Protopanaxadiol-Enriched Rice.

Event DS rice producing protopanaxadiol (PPD) has been previously developed by inserting Panax ginseng dammarenediol-II synthase gene (PgDDS) and PPD synthase gene (CYP716A47). We performed a gas chromatography-mass spectrometry (GC-MS)-based metabolomics of the DS rice to identify metabolic alterations as the effects of genetic engineering by measuring the contents of 65 metabolites in seeds and 63 metabolites in leaves. Multivariate analysis and one-way analysis of variance between DS and non-genetically modified (GM) rice showed that DS rice accumulated fewer tocotrienols, tocopherols, and phytosterols than non-GM rice. These results may be due to competition for the same precursors because PPDs in DS rice are synthesized from the same precursors as those of phytosterols. In addition, multivariate analysis of metabolic data from rice leaves revealed that composition differed by growth stage rather than genetic modifications. Our results demonstrate the potential of metabolomics for identifying metabolic alterations in response to genetic modifications.

Effects of Carbohydrates on Rosmarinic Acid Production and In Vitro Antimicrobial Activities in Hairy Root Cultures of Agastache rugosa.

Agastache rugosa (popularly known as Korean mint) belongs to the Lamiaceae family and comprises 22 species of perennial aromatic medicinal species native to East Asian countries, such as Korea, Taiwan, Japan, and China. A. rugosa contains many phenolic compounds that exhibit pharmacological and physiological activities, including antioxidant, anticancer, antiviral, antifungal, and antibacterial activities. The highest concentrations of rosmarinic acid and its isomers have been reported in the roots of A. rugosa. In this in vitro study, hairy roots of A. rugosa were obtained and the carbohydrates (sorbitol, mannitol, glucose, maltose, galactose, mannose, and sucrose) were evaluated to determine those that were optimal for rosmarinic acid production and hairy root growth. Antioxidant and antibacterial activities of extracts of A. rugosa were also assessed. The best carbon source for A. rugosa hairy root cultures was sucrose, considering biomass productivity (0.460 ± 0.034 mg/30 mL), rosmarinic acid production (7.656 ± 0.407 mg/g dry weight), and total phenolic content (12.714 ± 0.202 mg/g gallic acid equivalent). Antioxidant and antimicrobial activities were displayed by A. rugosa hairy roots cultured in liquid medium supplemented with 100 mM sucrose. Twenty-five bacterial strains, including multidrug-resistant bacteria and one pathogenic yeast strain, were used for antimicrobial screening of A. rugosa hairy roots. The hairy root extracts displayed antibacterial activity against Micrococcus luteus (KCTC 3063) and Bacillus cereus (KCTC 3624). The inhibition of these bacteria was greater using A. rugosa hairy roots with the highest levels of phenolic compounds cultured in the presence of sucrose, compared to hairy roots with the lowest levels of phenolic compounds cultured in the presence of fructose. Considering hairy root biomass, phenolic compound production, and antibacterial activity, sucrose is the best carbon source for A. rugosa hairy root cultures.