Overexpression of a cytosolic ascorbate peroxidase from Panax ginseng enhanced salt tolerance in Arabidopsis thaliana

Ascorbate peroxidase (APX) plays an essential role in the antioxidant defense mechanism in the plant, serving in the ascorbate–glutathione cycle for the cellular H2O2 metabolism process. As the perennial plant, Panax ginseng Meyer encounters a lot of unfavorable growth conditions, and among them soil salinity significantly decreases the yield. Two APX genes from Panax ginseng were isolated and designated as PgAPX1 and PgAPX2, which are most similar with previously characterized cytosolic APX of Daucus carota and Spuriopimpinella brachycarpa, as revealed by sequence analysis of their deduced amino acid sequences. PgAPXs transcripts are most abundant in leaf tissue, whereas PgAPX1 expression level was higher compared to PgAPX2. Consistent with higher PgAPX1 expression during salt stress in ginseng, PgAPX1-overexpressing Arabidopsis lines (PgAPX1ox) increased the germination rate and root length compared with wild-type (WT) under 200 mM NaCl stress treatment. Furthermore, higher chlorophyll content, relative water content, total APX activity, proline content, and lower H2O2 accumulation were shown in PgAPX1ox plants compared to WT under 200 mM NaCl stress condition. Transcription analysis showed that PgAPX1oxArabidopsis lines were more salt-tolerant by upregulating the ion homeostasis mechanism

Molecular Characterization and Expression Analysis of Pathogenesis related Protein 6 from Panax ginseng

Panax ginseng Meyer is one of the important medicinal plants in the world, particularly in Asian countries. Ginseng encounters many stress exposure during its long cultivation period. However, the molecular mechanism of stress resistance is still poorly understood in spite of its importance. In this study, pathogenesis- related protein 6 (PR6), also called proteinase inhibitor (PI), was isolated from ginseng embryogenic callus, named PgPR6. The small size of PR6, containing an open reading frame of 219 bp encoding 72 amino acids, the typical characteristic of PR6 protein, shares the highest sequence similarity to PR6 of Theobroma cacao (69% identity). Sequence and structural analysis indicated that PgPR6 belongs to class Kunitz-type PI family. This is the first report pertaining to the identification of PR6 gene from the P. ginseng genome. The high-level expression of PgPR6 was observed in root as revealed by quantitative real-time PCR. The temporal expression analysis demonstrated that PgPR6 expression was highly up-regulated by signaling molecules, heavy metals, mechanical wounding, chilling, salt, sucrose, and mannitol stress, indicating that PgPR6 may play an important role in the molecular defense response of ginseng to a various range of environmental stresses

Ectopic overexpression of the aluminum-induced protein gene from Panax ginseng enhances heavy metal tolerance in transgenic Arabidopsis

Aluminum (Al), one of the most abundant metals in agricultural soils, significantly limits crop growth and productivity in acidic soil by inhibiting root elongation. Al ions, especially Al3+, have a toxic effect on both plant and animal cells under low-pH conditions. We first isolated and characterized aluminum-induced protein (AIP) cDNA from a 4-year-old root of Panax ginseng Meyer. This cDNA encodes an open reading frame of 711 bp with a deduced amino acid sequence of 236 residues. The calculated molecular mass of the mature protein is approximately 58.9 kDa with a predicated isoelectric point of 5.13. The Panax ginsengAIP (PgAIP) contains a domain also present in wheat aluminum-induced protein 7 (Wali7) and shares homology with the AIPs of other species, including Codonopis and Arabidopsis. The PgAIP gene was abundantly expressed in the plant’s leaves and was up-regulated by Al exposure. The functional role of PgAIP in Al tolerance was further validated through its overexpression in Arabidopsis. Transgenic Arabidopsis plants overexpressing the PgAIP gene showed enhanced Al tolerance in terms of root growth when compared to wild-type plants, suggesting PgAIP is important in plant defense against Al toxicity. Confocal analysis of CFP-tagging PgAIP in Arabidopsis showed subcellular localization in the plasma membrane. Our results suggest that PgAIP in the plasma membrane plays an important role in the protection of plant cells against heavy metal exposure

Functional characterization of the pathogenesis-related protein family 10 gene, PgPR10-4, from Panax ginseng in response to environmental stresses

Pathogenesis-related proteins (PRs) are known to function in higher plants as a protein-based defensive system against abiotic and biotic stress, particularly pathogen infections. A full-length cDNA sequence of PR BetV1 was isolated and characterized from a 14-year-old ginseng expressed sequence tags library and we named this as PgPR10-4, because of similar identities with previous isolated PgPR10s sequences. The PgPR10-4 gene encodes a 477 bp open reading frame and its deduced protein contains 158 amino acids with a 53 % identity with that of the Actinidia chinensis BetV1 allergen. The expression of PgPR10-4 gene was abundant in leaves and its transcripts showed differentially up-regulated patterns against several ginseng pathogens and abiotic stimuli such as high light and salinity. In addition, PgPR10-4 expression was strongly responsive towards the stress signaling molecules H2O2 and jasmonic acid (JA), while weekly responsive to salicylic acid and abscisic acid. A functional role of PgPR10-4 in environmental stress tolerance was further validated through its overexpression in Arabidopsis. An analysis of T2 transgenic Arabidopsis plants overexpressing the PgPR10-4 gene showed an enhanced tolerance to bacterial and fungal infection, but not to salt stress. When we tagged with cyan fluorescent protein fusion protein, the PgPR10-4-was found to localize to the cytoplasm. The enhanced antifungal activity observed from the Arabidopsis transgenic lines suggests the possible involvement of PgPR10-4 in a defense-related mechanism via the JA signaling pathway

Cytological analysis of ginseng carpel development

Panax ginseng Meyer, commonly known as ginseng, is considered one of the most important herbs with pharmaceutical values due to the presence of ginsenosides and is cultivated for its highly valued root for medicinal purposes. Recently, it has been recognized that ginseng fruit contains high contents of triterpene such as ginsenoside Re as pharmaceutical compounds. However, it is unclear how carpel, the female reproductive tissue of flowers, is formed during the three-year-old growth before fruit is formed in ginseng plants. Here, we report P. ginseng carpel development at the cytological level, starting from the initial stage of ovule development to seed development. The carpel of P. ginseng is composed of two free stigmas, two free styles, and one epigynous bilocular ovary containing one ovule in each locule. Based on our cytological study, we propose that the female reproductive development in P. ginseng can be classified into seven stages: early phase of ovule development, megasporogenesis, megagametogenesis, pre-fertilization, fertilization, post-fertilization, and seed development. We also describe the correlation of the female and male gametophyte development and compare morphological differences in carpel development between ginseng and other higher plants. One unique feature for ginseng seed development is that it takes 40 days for the embryo to develop to the early torpedo stage and that the embryo is small relative to the seed size, which could be a feature of taxonomic importance. This study will provide an integral tool for the study of the reproductive development and breeding of P. ginseng

Molecular characterization of two glutathione peroxidase genes of Panax ginseng and their expression analysis against environmental stresses

Glutathione peroxidases (GPXs) are a group of enzymes that protect cells against oxidative damage generated by reactive oxygen species (ROS). GPX catalyzes the reduction of hydrogen peroxide (H2O2) or organic hydroperoxides to water or alcohols by reduced glutathione. The presence of GPXs in plants has been reported by several groups, but the roles of individual members of this family in a single plant species have not been studied. Two GPX cDNAs were isolated and characterized from the embryogenic callus of Panax ginseng. The two cDNAs had an open reading frame (ORF) of 723 and 681 bp with a deduced amino acid sequence of 240 and 226 residues, respectively. The calculated molecular mass of the matured proteins are approximately 26.4 kDa or 25.7 kDa with a predicated isoelectric point of 9.16 or 6.11, respectively. The two PgGPXs were elevated strongly by salt stress and chilling stress in a ginseng seedling. In addition, the two PgGPXs showed different responses against biotic stress. The positive responses of PgGPX to the environmental stimuli suggested that ginseng GPX may help to protect against environmental stresses

Development of EST Intron-Targeting SNP Markers for Panax ginseng and Their Application to Cultivar Authentication

Panax ginseng is one of the most valuable medicinal plants in the Orient. The low level of genetic variation has limited the application of molecular markers for cultivar authentication and marker-assisted selection in cultivated ginseng. To exploit DNA polymorphism within ginseng cultivars, ginseng expressed sequence tags (ESTs) were searched against the potential intron polymorphism (PIP) database to predict the positions of introns. Intron-flanking primers were then designed in conserved exon regions and used to amplify across the more variable introns. Sequencing results showed that single nucleotide polymorphisms (SNPs), as well as indels, were detected in four EST-derived introns, and SNP markers specific to “Gopoong” and “K-1” were first reported in this study. Based on cultivar-specific SNP sites, allele-specific polymerase chain reaction (PCR) was conducted and proved to be effective for the authentication of ginseng cultivars. Additionally, the combination of a simple NaOH-Tris DNA isolation method and real-time allele-specific PCR assay enabled the high throughput selection of cultivars from ginseng fields. The established real-time allele-specific PCR assay should be applied to molecular authentication and marker assisted selection of P. ginseng cultivars, and the EST intron-targeting strategy will provide a potential approach for marker development in species without whole genomic DNA sequence information

Development of a single-nucleotide-polymorphism marker for specific authentication of Korean ginseng (Panax ginseng Meyer) new cultivar “G-1”

Background: Korean ginseng (Panax ginseng) is a well-known medicinal plant of Oriental medicine that is still in practice today. Until now, a total of 11 Korean ginseng cultivars with unique features to Korean ginseng have been developed based on the pure-line-selection method. Among them, a new cultivar namely G-1 with different agricultural traits related to yield and content of ginsenosides, was developed in 2012. Methods: The aim of this study was to distinguish the new ginseng cultivar G-1 by identifying the unique single-nucleotide polymorphism (SNP) at its 45S ribosomal DNA and Panax quinquefolius region than other Korean ginseng cultivars using multiplex amplification-refractory mutation system–polymerase chain reaction (ARMS-PCR). Results: A SNP at position of 45S ribosomal DNA region between G-1, P. quinquefolius, and the other Korean ginseng cultivars was identified. By designing modified allele-specific primers based on this site, we could specifically identified G-1 and P. quinquefolius via multiplex PCR. The unique primer for the SNP yielded an amplicon of size 449 bp in G-1 cultivar and P. quinquefolius. This study presents an effective method for the genetic identification of the G-1 cultivar and P. quinquefolius. Conclusion: The results from our study shows that this SNP-based approach to identify the G-1 cultivar will be a good way to distinguish accurately the G-1 cultivar and P. quinquefolius from other Korean ginseng cultivars using a SNP at 45S ribosomal DNA region