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Programmed cell death (PCD), a characteristic feature of hypersensitive response (HR) in plants, is an important cellular process often associated with the defense response against pathogens. Here, the involvement of LytB, a gene encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase that participates in the final step of the plastid methylerythritol phosphate (MEP) pathway, in plant HR cell death was studied. In Nicotiana benthmiana plants, silencing of the NbLytB gene using virus-induced gene silencing (VIGS) caused plant growth retardation and albino leaves with severely malformed chloroplasts. In NbLytB-silenced plants, HR-related cell death mediated by the expression of either the human proapoptotic protein gene Bax or an R gene with its cognate Avr effector gene was inhibited, whereas that induced by the nonhost pathogen Pseudomonas syringae pv. syringae 61 was enhanced. To dissect the isoprenoid pathway and avoid the pleiotropic effects of VIGS, chemical inhibitors that specifically inhibit isoprenoid biosynthesis in plants were employed. Treatment of N. benthamiana plants with fosmidomycin, a specific inhibitor of the plastid MEP pathway, effectively inhibited HR-related PCD, whereas treatment with mevinolin (a cytoplasmic mevalonate pathway inhibitor) and fluridone (a carotenoid biosynthesis inhibitor) did not. Together, these results suggest that the MEP pathway as well as reactive oxygen species (ROS) generation in the chloroplast play an important role in HR-related PCD, which is not displaced by the cytosolic isoprenoid biosynthesis pathway.

Ginseng (Panax ginseng) has a low genetic diversity and a narrow pool of genetic resources. Mutagenesis is one of the most powerful methods for inducing genetic variation in this species, but little research has been performed in ginseng. In this study, various tissues, including dehiscent seeds, 1-year-old roots, and somatic embryos were irradiated at different doses of gamma rays (20–400 Gy and zero dose as a control) to determine the most optimal concentration and tissues for the successful use of mutagenesis in ginseng breeding. The results revealed that high gamma doses (> 100 Gy) were detrimental to all irradiated tissues. A gradual and significant reduction in germination, emergence, and seedling growth were found as gamma irradiation dose increased. The reduction in survival rates and seedling growth by irradiation at serial doses showed that the LD50 of ginseng was 20–80 Gy, although the irradiation doses were tissue dependent. Based on our results, the optimal doses of gamma rays for inducing mutation in ginseng are

Background: Ginseng is one of the well-known medicinal plants, exhibiting diverse medicinal effects. Its roots possess anticancer and antiaging properties and are being used in the medical systems of East Asian countries. It is grown in low-light and low-temperature conditions, and its growth is strongly inhibited at temperatures above 25°C. However, the molecular responses of ginseng to heat stress are currently poorly understood, especially at the protein level. Methods: We used a shotgun proteomics approach to investigate the effect of heat stress on ginseng leaves. We monitored their photosynthetic efficiency to confirm physiological responses to a high-temperature stress. Results: The results showed a reduction in photosynthetic efficiency on heat treatment (35°C) starting at 48 h. Label-free quantitative proteome analysis led to the identification of 3,332 proteins, of which 847 were differentially modulated in response to heat stress. The MapMan analysis showed that the proteins with increased abundance were mainly associated with antioxidant and translation-regulating activities, whereas the proteins related to the receptor and structural-binding activities exhibited decreased abundance. Several other proteins including chaperones, G-proteins, calcium-signaling proteins, transcription factors, and transfer/carrier proteins were specifically downregulated. Conclusion: These results increase our understanding of heat stress responses in the leaves of ginseng at the protein level, for the first time providing a resource for the scientific community. Keywords: High temperature, Panax ginseng, Photosynthetic efficiency, Shotgun proteomics

Panax ginseng C. A. Mey., a perennial herb belonging to the family Araliaceae, is a valuable medicinal plant with distinctive biological characteristics. However, comprehensive analyses of the mitochondrial genome (mitogenome) are lacking. In this study, we sequenced the complete mitogenome of ginseng based on long-read data from the Nanopore sequencing platform. The mitogenome was assembled into a &ldquo, master circle&rdquo, form of 464,705 bp and contained 72 unique genes. The genome had three large repeat regions, and 10.42% of the sequences were mitogenome sequences of plastid origin (MTPTs). In total, 278 variants (213 SNPs and 65 InDels) were discovered, most of which were identified in intergenic regions. The MTPT regions were mutational hotspots, harboring 74.5% of the variants. The ginseng mitogenome showed a higher mutation rate than that of the chloroplast genome, and this pattern is uncommon in plants. In addition, 10 Kompetitive allele-specific PCR (KASP) markers were developed from 10 SNPs, excluding those in MTPT regions. These markers accurately identified the genotypes of 59 Korean ginseng accessions and elucidated mitogenome diversity. These results provide insight into organellar genomes and genetic diversity in ginseng. Moreover, the complete mitogenome sequence and 10 KASP markers will be useful for ginseng research and breeding.

Ginseng (Panax ginseng C.A. Meyer) is a medicinal crop that requires a long culture time before it is ready to harvest, thus generating high economic and environmental costs. Symbiotic bacteria that live within the plant provide the host plant with many advantages in terms of metabolism and disease resistance. Here, we isolated endophytic bacteria from various tissues of P. ginseng seedlings using a culture-dependent method and we compared their tissue distribution. In addition, their antimicrobial activity against two fungal pathogens was investigated. Based on 16S rRNA sequencing, we identified 21 bacterial strains from ginseng seedlings. Leaves and rhizomes showed higher bacterial species diversity than root bodies and tails. While Bacillus strains were detected in all tissues, Xanthomonas and Micrococcaceae strains were specifically isolated from rhizome and leaf tissues, respectively. Fourteen bacterial strains showed antimicrobial activities against Cylindrocarpon destructans and/or Botrytis cinerea, with different activities. Among them, two strains (PgKB29 and PgKB35) showed strong antimicrobial activities against both fungi. Taken together, these results provide a better understanding of endophytic bacteria in P. ginseng seedlings and suggest the possibility of biological control of fungal pathogens using endophytic bacteria.

Korean ginseng (Panax ginseng C.A. Meyer) is a largely sought after resource due to its substantial health benefits. However, large-scale culture of ginseng seeds is limited because of its long maturation time and triple-dormancy. To develop new strategies to improve the dehiscence, germination, and breaking dormancy, we investigated the effects of stratification, gibberellin (GA3), and/or kinetin treatments on the dehiscence, development and germination of P. ginseng. Indehiscent seeds for the dehiscence test and dehiscent seeds for the germination test were immersed in a solution containing 288.7 μM GA3 and/or 232.3 μM kinetin for 24 h, respectively, at 25 °C. After dehiscence, a cold stratification experiment was conducted at 2 °C for 15, 30, 45, 60, and 90 days in the dark. Exogenous GA3 and kinetin treatments significantly improved the dehiscence rate. In addition, both plant growth regulators (PGRs) appeared to enhance the zygotic embryo development of ginseng seeds. GA3 on its own had a slight effect on breaking dormancy and germination, but the effect was not sufficient to serve as a complete substitute for the cold stratification requirement due to the deep physiological dormancy characteristic of P. ginseng. Kinetin treatment had a significant effect on germination and breaking dormancy, although a short period of cold stratification was also required. The combination of GA3 and kinetin treatments was more effective at enhancing both seed dehiscence and germination than either PGR treatment alone.

Despite many studies on tissue culture of Panax ginseng, the propagation of cultivars is still dependent on seeds due to low survival rate of plants produced in vitro. Healthy in vitro grown plants guarantee high survival rates after they are transferred to soil, but limited information exists on the optimum medium for the in vitro growth of P. ginseng. In this study, zygotic embryo culture was used to develop an optimum medium suitable for in vitro cultivation of P. ginseng. After 4–6 months of culture, the aged aerial parts turned yellow, and the in vitro grown roots (IGRs) had thickened taproots with dormant rhizomes. The seedling growth was evaluated using various concentrations of sucrose and basal media at various strengths. Of the tested media, 2% sucrose and 1/3 strength SH medium showed superior results. Subsequently, IGRs were transferred to soil and evaluated for their morpho-anatomical characteristics. Furthermore, their plants were analyzed for genetic fidelity using molecular markers. IGRs showed outstanding morphological differences compared with conventionally grown 1-year-old ginseng roots; however, there was no difference in the sprouting and survival rate after soil transfer. 2-year-old IGRs deprived from the IGRs have intermediate properties between 1-year-old and 2-year-old root. Unlike the 2-year-old roots, the morphology of 2-year-old IGRs had shorter primary roots with several lateral roots. Despite the morpho-anatomical differences, molecular marker analysis also confirmed that the IGRs had genetic fidelity.

This study investigated the root-associated bacterial endophytes of Panax ginseng at different ages by shotgun metagenomic analysis. After mapping metagenome data to the complete ginseng genome to identify unmapped sequences, we predicted the structure and functions of ginseng bacterial endophytes by metagenomic rapid annotation using subsystems technology analysis. While Proteobacteria and Actinobacteria were the predominant phyla in all samples (2–6-year-old roots), class Alphaproteobacteria was most abundant in 3-, 4-, and 5-year-old plants. We found that 3-year-old P. ginseng had a 0.66% unmapped rate against the whole ginseng genome and showed the greatest diversity of endophytic bacteria (α diversity = 299). Prediction of endophytic bacterial functions at different ages by SEED subsystem analysis revealed that siderophore and auxin-related traits—which are known to promote plant growth—were most highly represented in 3-year-old plants. This was supported by a gene frequency analysis of plant growth-promoting genes, including those responsible for solubilization of phosphate and nitrogen metabolism, using BLASTn. These results suggest that endophytic bacteria of the P. ginseng root affect plant growth. Furthermore, the isolation and purification of plant growth-promoting endophytes identified in this study could promote sustainable cultivation of ginseng in the future.

Background Licorice (Glycyrrhiza spp. L.) is used as a natural sweetener and medicinal herb in European and Asian countries. Molecular studies have been conducted to find differences between wild and cultivated species because most wild species are highly resistant to abiotic and biotic stresses compared with their cultivated species. However, few molecular markers have been developed for studying the genetic diversity and population structure of licorice species and to identify differences between cultivars. Thus, the present study aimed to develop a set of genomic simple sequence repeat (SSR) markers for molecular studies of these species. Methods In the present study, we developed polymorphic SSR markers based on whole-genomesequence data of Glycyrrhiza lepidota. Then, based on the sequence information, the polymorphic SSR markers were developed. The SSR markers were applied to 23 Glycyrrhiza individual plants. We also evaluated the phylogenetic relationships and interspecies transferability among samples. Results The genetic diversity analysis using these markers identified 2–23 alleles, and the major allele frequency, observed heterozygosity, genetic diversity, and polymorphism information content were 0.11–0.91, 0–0.90, 0.17–0.94, and 0.15–0.93, respectively. Interspecies transferability values were 93.5%, 91.6%, and 91.1% for G. echinata, G. glabra, and G. uralensis, respectively. Phylogenetic analysis clustered cultivated (group 1) and wild (group 2) species into three and two subgroups, respectively. The reported markers represent a valuable resource for the genetic characteri z ation of Glycyrrhiza spp. for theanalysis of its genetic variability, and as a tool for licorice transferability. This is the first intraspecific study in a collection of Glycyrrhiza spp. germplasm using SSR markers.

Most plants co-exist with fungal and bacterial endophytes. Generally, endophytes are beneficial microorganisms that colonize the internal tissues of their host plants. Plants derive several advantages from endophytic colonization, such as the biocontrol of phytopathogens and growth-promoting factors. In this review, we discuss the current knowledge of endophytic bacteria and their potential as natural biocontrol agents for plants.

Rhodococcus species have become increasingly important owing to their ability to degrade a wide range of toxic chemicals and produce bioactive compounds. Here, we report isolation of the Rhodococcus sp. KB6, which is a new leaf-inhabiting endophytic bacterium that suppresses black rot disease in sweet potato leaves. We determined the 7.0 Mb draft genome sequence of KB6 and have predicted 19 biosynthetic gene clusters for secondary metabolites, including heterobactins, which are a new class of siderophores. Notably, we showed the first internal colonization of host plants with Rhodococcus sp. KB6 and discuss its potential as a biocontrol agent for sustainable agriculture.

Bacillus cereus PgBE311, isolated from the root tissue of a 5-year-old Panax ginseng plant, showed activities against the fungal pathogens Cylindrocarpon destructans and Botrytis cinerea . Here, we report the genome sequence of B. cereus PgBE311.

Bacillus cereus PgBE311, isolated from the root tissue of a 5-year-old Panax ginseng plant, showed activities against the fungal pathogens Cylindrocarpon destructans and Botrytis cinerea. Here, we report the genome sequence of B. cereus PgBE311., Bacillus cereus PgBE311, isolated from the root tissue of a 5-year-old Panax ginseng plant, showed activities against the fungal pathogens Cylindrocarpon destructans and Botrytis cinerea. Here, we report the genome sequence of B. cereus PgBE311. The bacterium contains antibiotic-related gene clusters and has the potential to stimulate plant growth.

Chryseobacterium indologenes PgBE177, isolated from the root tissue of a 4-year-old Panax quinquefolius plant, showed antagonistic activity against Pseudomonas syringae pv. tomato DC3000, a bacterial pathogen., Chryseobacterium indologenes PgBE177, isolated from the root tissue of a 4-year-old Panax quinquefolius plant, showed antagonistic activity against Pseudomonas syringae pv. tomato DC3000, a bacterial pathogen. Here, we report the whole-genome sequence of C. indologenes PgBE177. The bacterium contains bacteriocin gene clusters and has the potential to stimulate plant growth.

Although endophytic bacteria are known to colonize Panax ginseng, little is known about their diversity and roles. We addressed in the present study by comparing endophytic bacterial populations in P. ginseng plants of different ages (2–6 years) and in various tissue types (root, stem, leaf, and flower stalk). A total of 116 strains assigned to 42 species were identified by 16S rDNA sequencing. The predominant phylum was Firmicutes. Two-year-old ginseng plants and root tissues showed the greatest diversity of endophytic bacteria, with Bacillales being the predominant order. The antifungal activity of isolates against two pathogens, Cylindrocarpon destructans and/or Botrytis cinerea, was evaluated in dual-culture assays. In total, 28 strains showed antifungal activity with PgBE14 (Bacillus amyloliquefaciens), PgBE40 (B. megaterium), PgBE39, PgBE45 (Pseudomonas frederiksbergensis), and PgBE42 (Staphylococcus saprophyticus) inhibiting both pathogens. These results improve our understanding of the structure and diversity of endophytic bacterial communities of P. ginseng and identify strains with antifungal activity that have potential applications as biocontrol agents.

Ginseng is one of the well-known medicinal plants, exhibiting diverse medicinal effects. Its roots possess anticancer and antiaging properties and are being used in the medical systems of East Asian countries. It is grown in low-light and low-temperature conditions, and its growth is strongly inhibited at temperatures above 25°C. However, the molecular responses of ginseng to heat stress are currently poorly understood, especially at the protein level.We used a shotgun proteomics approach to investigate the effect of heat stress on ginseng leaves. We monitored their photosynthetic efficiency to confirm physiological responses to a high-temperature stress.The results showed a reduction in photosynthetic efficiency on heat treatment (35°C) starting at 48 h. Label-free quantitative proteome analysis led to the identification of 3,332 proteins, of which 847 were differentially modulated in response to heat stress. The MapMan analysis showed that the proteins with increased abundance were mainly associated with antioxidant and translation-regulating activities, whereas the proteins related to the receptor and structural-binding activities exhibited decreased abundance. Several other proteins including chaperones, G-proteins, calcium-signaling proteins, transcription factors, and transfer/carrier proteins were specifically downregulated.These results increase our understanding of heat stress responses in the leaves of ginseng at the protein level, for the first time providing a resource for the scientific community.

Licorice (Glycyrrhiza glabra) is an important medicinal crop often used as health foods or medicine worldwide. The molecular genetics of licorice is under scarce owing to lack of molecular markers. Here, we have developed cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers based on single nucleotide polymorphisms (SNP) by comparing the chloroplast genomes of two Glycyrrhiza species (G. glabra and G. lepidota). The CAPS and HRM markers were tested for diversity analysis with 24 Glycyrrhiza accessions. The restriction profiles generated with CAPS markers classified the accessions (2-4 genotypes) and melting curves (2-3) were obtained from the HRM markers. The number of alleles and major allele frequency were 2-6 and 0.31-0.92, respectively. The genetic distance and polymorphism information content values were 0.16-0.76 and 0.15-0.72, respectively. The phylogenetic relationships among the 24 accessions were estimated using a dendrogram, which classified them into four clades. Except clade III, the remaining three clades included the same species, confirming interspecies genetic correlation. These 18 CAPS and HRM markers might be helpful for genetic diversity assessment and rapid identification of licorice species.

Licorice (Glycyrrhiza glabra) is an important medicinal crop often used as health foods or medicine worldwide. The molecular genetics of licorice is under scarce owing to lack of molecular markers. Here, we have developed cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers based on single nucleotide polymorphisms (SNP) by comparing the chloroplast genomes of two Glycyrrhiza species (G. glabra and G. lepidota). The CAPS and HRM markers were tested for diversity analysis with 24 Glycyrrhiza accessions. The restriction profiles generated with CAPS markers classified the accessions (2–4 genotypes) and melting curves (2–3) were obtained from the HRM markers. The number of alleles and major allele frequency were 2−6 and 0.31–0.92, respectively. The genetic distance and polymorphism information content values were 0.16–0.76 and 0.15–0.72, respectively. The phylogenetic relationships among the 24 accessions were estimated using a dendrogram, which classified them into four clades. Except clade III, the remaining three clades included the same species, confirming interspecies genetic correlation. These 18 CAPS and HRM markers might be helpful for genetic diversity assessment and rapid identification of licorice species.

Bacterial endophytes benefit the host with protection against abiotic and biotic stresses and through increased plant growth. In this study, we screened novel endophytes for the ability to endophytically inhabit leaf tissues. Four endophytic bacteria were screened from the apoplastic fluids extracted from the uninfected upper leaves of Arabidopsis thaliana 3 days postinoculation (dpi) with Pseudomonas syringae pv. tomato DC3000 (Pst). Under sterile plant growth conditions, we showed that the four isolates proliferated intercellularly in the leaf tissues of A. thaliana. Based on 16S rRNA sequencing analysis, the identities of the inoculated endophytes and the re-isolates from the leaf tissues were confirmed. Among the isolates, the two species of Rhodococcus were the first members of the genus to be identified as leaf-inhabiting endophytes. Additionally, three of the isolates showed antagonistic activities, with different levels of activity, against Fusarium oxysporum pv. conglutinans (F.o.) and/or Pst. Furthermore, the application of one isolate, Bacillus cereus KB1, on tomato plants significantly increased the disease resistance to Botrytis cinerea and Pst. In combination, these results indicate that these endophytic isolates can be used to develop potential biocontrol agents against a variety of pathogenic fungi and bacteria.

Korean ginseng (Panax ginseng C.A. Meyer) has been widely used for medicinal purposes and contains potent plant secondary metabolites, including ginsenosides. To obtain transcriptomic data that offers a more comprehensive view of functional genomics in P. ginseng, we generated genome-wide transcriptome data from four different P. ginseng tissues using PacBio isoform sequencing (Iso-Seq) technology. A total of 135,317 assembled transcripts were generated with an average length of 3.2 kb and high assembly completeness. Of those unigenes, 67.5% were predicted to be complete full-length (FL) open reading frames (ORFs) and exhibited a high gene annotation rate. Furthermore, we successfully identified unique full-length genes involved in triterpenoid saponin synthesis and plant hormonal signaling pathways, including auxin and cytokinin. Studies on the functional genomics of P. ginseng seedlings have confirmed the rapid upregulation of negative feed-back loops by auxin and cytokinin signaling cues. The conserved evolutionary mechanisms in the auxin and cytokinin canonical signaling pathways of P. ginseng are more complex than those in Arabidopsis thaliana. Our analysis also revealed a more detailed view of transcriptome-wide alternative isoforms for 88 genes. Finally, transposable elements (TEs) were also identified, suggesting transcriptional activity of TEs in P. ginseng. In conclusion, our results suggest that long-read, full-length or partial-unigene data with high-quality assemblies are invaluable resources as transcriptomic references in P. ginseng and can be used for comparative analyses in closely related medicinal plants.

Variovorax paradoxus KB5, isolated from the inside of Arabidopsis thaliana leaves, showed antibacterial activity against the phytopathogen Pseudomonas syringae pv. tomato DC3000. Here, we report a draft genome sequence of V. paradoxus KB5, which contains a delftibactin-like nonribosomal peptide biosynthetic gene cluster.

Rhodococcus kyotonensis KB10 is an endophytic bacterium isolated from Arabidopsis thaliana . The organism showed mild antibacterial activity against the phytopathogen Pseudomonas syringae pv. tomato DC3000. This study reports the genome sequence of R. kyotonensis KB10. This bacterium contains an ectoine biosynthesis gene cluster and has the potential to degrade nitroaromatic compounds. The identified bacterium may be a suitable biocontrol agent and degrader of environmental pollutants.

Alpha-dioxygenases (α-DOX) catalyzing the primary oxygenation of fatty acids to oxylipins were recently found in plants. Here, the biological roles of the pepper α-DOX (Ca-DOX) gene, which is strongly induced during non-host pathogen infection in chili pepper, were examined. Virus-induced gene silencing demonstrated that down-regulation of Ca-DOX enhanced susceptibility to bacterial pathogens and suppressed the hypersensitive response via the suppression of pathogenesis-related genes such as PR4, proteinase inhibitor II and lipid transfer protein (PR14). Ca-DOX-silenced pepper plants also exhibited more retarded growth with lower epidermal cell numbers and reduced cell wall thickness than control plants. To better understand regulation of Ca-DOX, transgenic Arabidopsis plants harboring the β-glucuronidase (GUS) reporter gene driven from a putative Ca-DOX promoter were generated. GUS expression was significantly induced upon avirulent pathogen infection in transgenic Arabidopsis leaves, whereas GUS induction was relatively weak upon virulent pathogen treatment. After treatment with plant hormones, early and strong GUS expression was seen after treatment of salicylic acid, whereas ethylene and methyl jasmonate treatments produced relatively weak and late GUS signals. These results will enable us to further understand the role of α-DOX, which is important in lipid metabolism, defense responses, and growth development in plants.

Paenibacillus polymyxa AC-1 (AC-1) is a plant growth-promoting rhizobacterium (PGPR) that has been used as a soil inoculant for biocontrol of plant pathogenic fungi and to promote plant growth. In this study, we examine the effects of AC-1 on the bacterial phytopathogen Pseudomonas syringae and internal colonization of AC-1 by counting bacterial populations that colonize plants. AC-1 inhibited the growth of both P. syringae pv. tomato DC3000 (Pst) and P. syringae pv. tabaci (Pta) in a concentration-dependent manner in in vitro assays. Upon treatment of AC-1 dropping at root tip of axenically grown Arabidopsis, we found that most of the AC-1 was detected in interior of leaves of Arabidiopsis plants rather than roots after 5 days post infection, indicating systemic spreading of AC-1 occur. We examined further AC-1 colonization patterns in Arabidopsis mutants deficient in phytohormone signaling pathways. These results indicated that abscisic acid (ABA) and jasmonic acid (JA) signaling pathways positively and negatively contributed, respectively, to AC-1 colonization of leaves, whereas epiphytic accumulation of AC-1 around root tissues was not affected. This study shows that AC-1 is an effective biocontrol agent to suppress P. syringae growth, possibly owing to its colonization patterns as a leaf-inhabiting endophyte. The results showed in this work will help to expand our understanding of the mode of action of AC-1 as a biological control agent and consequently, its application in agriculture.