Yurry Um

Global Profiling of Various Metabolites in Platycodon grandiflorum by UPLC-QTOF/MS

In this study, a method of metabolite profiling based on UPLC-QTOF/MS was developed to analyze Platycodon grandiflorum. In the optimal UPLC, various metabolites, including major platycosides, were separated well in 15 min. The metabolite extraction protocols were also optimized by selecting a solvent for use in the study, the ratio of solvent to sample and sonication time. This method was used to profile two different parts of P. grandiflorum, i.e., the roots of P. grandiflorum (PR) and the stems and leaves of P. grandiflorum (PS), in the positive and negative ion modes. As a result, PR and PS showed qualitatively and quantitatively different metabolite profiles. Furthermore, their metabolite compositions differed according to individual plant samples. These results indicate that the UPLC-QTOF/MS-based profiling method is a good tool to analyze various metabolites in P. grandiflorum. This metabolomics approach can also be applied to evaluate the overall quality of P. grandiflorum, as well as to discriminate the cultivars for the medicinal plant industry.

Development of Genome-Wide SSR Markers from Angelica gigas Nakai Using Next Generation Sequencing

Angelica gigas Nakai is an important medicinal herb, widely utilized in Asian countries especially in Korea, Japan, and China. Although it is a vital medicinal herb, the lack of sequencing data and efficient molecular markers has limited the application of a genetic approach for horticultural improvements. Simple sequence repeats (SSRs) are universally accepted molecular markers for population structure study. In this study, we found over 130,000 SSRs, ranging from di- to deca-nucleotide motifs, using the genome sequence of Manchu variety (MV) of A. gigas, derived from next generation sequencing (NGS). From the putative SSR regions identified, a total of 16,496 primer sets were successfully designed. Among them, we selected 848 SSR markers that showed polymorphism from in silico analysis and contained tri- to hexa-nucleotide motifs. We tested 36 SSR primer sets for polymorphism in 16 A. gigas accessions. The average polymorphism information content (PIC) was 0.69; the average observed heterozygosity (HO) values, and the expected heterozygosity (HE) values were 0.53 and 0.73, respectively. These newly developed SSR markers would be useful tools for molecular genetics, genotype identification, genetic mapping, molecular breeding, and studying species relationships of the Angelica genus.

Identification of Endophytic Bacteria in Panax ginseng Seeds and Their Potential for Plant Growth Promotion

Endophytes are microorganisms that live in the internal tissues of plants without harming the host plants. In this symbiotic relationship, the host plants provide nutrients and shelter to the endophytes, in turn, endophytes can promote the growth of host plants and act as a biological control agents against plant pathogens. Plant-microbe interactions like this are noted for natural methods for sustainable agriculture and environmental conservation. However, in spite of the infinite potential, there are only a few reports on the endophytes present in ginseng. In this study, we isolated and identified the endophytes from Panax ginseng seeds and evaluated the biological activities (IAA production ability, nitrogen fixation ability, phosphate solubilization capacity, siderophore production ability, and antifungal activities) of the endophyte isolates. Eight different endophytes were identified by 16S rRNA sequencing. Most of the endophytes have antibiotic and plant growth promoting (PGP) activities. Particularly, PgSEB5-37E have the highest antibiotic activity, both PgSEB5-37B and PgSEB5-37H have high PGP traits such as an abilities to produce IAA, solubilize phosphate and fix nitrogen. These results indicated that the endophytes from P. ginseng seeds may have applicable value to many industries. In order to use the isolated endophytes, quantitative analysis and field tests are needed to be performed.

A Novel Multifunctional C-23 Oxidase, CYP714E19, is Involved in Asiaticoside Biosynthesis

Centella asiatica is widely used as a medicinal plant due to accumulation of the ursane-type triterpene saponins asiaticoside and madecassoside. The molecular structure of both compounds suggests that they are biosynthesized from α-amyrin via three hydroxylations, and the respective Cyt P450-dependent monooxygenases (P450 enzymes) oxidizing the C-28 and C-2α positions have been reported. However, a third enzyme hydroxylating C-23 remained elusive. We previously identified 40,064 unique sequences in the transcriptome of C. asiatica elicited by methyl jasmonate, and among them we have now found 149 unigenes encoding putative P450 enzymes. In this set, 23 full-length cDNAs were recognized, 13 of which belonged to P450 subfamilies previously implicated in secondary metabolism. Four of these genes were highly expressed in response to jasmonate treatment, especially in leaves, in accordance with the accumulation patterns of asiaticoside. The functions of these candidate genes were tested using heterologous expression in yeast cells. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that yeast expressing only the oxidosqualene synthase CaDDS produced the asiaticoside precursor α-amyrin (along with its isomer β-amyrin), while yeast co-expressing CaDDS and CYP716A83 also contained ursolic acid along with oleanolic acid. This P450 enzyme thus acts as a multifunctional triterpenoid C-28 oxidase converting amyrins into corresponding triterpenoid acids. Finally, yeast strains co-expressing CaDDS, CYP716A83 and CYP714E19 produced hederagenin and 23-hydroxyursolic acid, showing that CYP714E19 is a multifunctional triterpenoid oxidase catalyzing the C-23 hydroxylation of oleanolic acid and ursolic acid. Overall, our results demonstrate that CaDDS, CYP716A83 and CYP714E19 are C. asiatica enzymes catalyzing consecutive steps in asiaticoside biosynthesis.

Morphological Characteristics and Genetic Diversity Analysis of Platycodon grandiflorum (Jacq.) A. DC Determined Using SSR Markers

Background : Plant breeding requires the collection of genetically diverse genetic resources. Studies on the characteristics of Platycodon grandiflorum resources have not been carried out so far. The present study was carried out to discriminate P. grandiflorum based on morphological characteristics and genetic diversity using simple sequence repeat (SSR) markers. Methods and Results :We collected 11 P. grandiflorum cultivars: Maries II, Hakone double white, Hakone double blue, Fuji white, Fuji pink, Fuji blue, Astra white, Astra pink, Astra blue, Astra semi-double blue and Jangbaek. Analyses of the morphological characteristics of the collection were conducted for aerial parts (flower, stem and leaf) and underground parts (root). Next, the genetic diversity of all P. grandiflorum resources was analyzed using SSR markers employing the DNA fragment analysis method. We determined that the 11 P. grandiflorum cultivars analyzed could be classified by plant length, leaf number and root characteristic. Based on the genetic diversity analysis, these cultivars were classified into four distinct groups. Conclusions : These findings could be used for further research on cultivar development using molecular breeding techniques and for conservation of the genetic diversity of P. grandiflorum. Moreover, the markers could be used for genetic mapping of the plant and marker-assisted selection for crop breeding.

Production of Adventitious Root and Analysis of Effective Components from in vitro Culture of Astragalus membranaceus

Background : A series of studies were conducted to optimize adventitious root induction in vitro from explants of Astragalus membranaceus using various nutrient media supplemented with plant hormones. Methods and Results : Levels of active components were analyzed from adventitious roots induced under different media conditions. Among the different media conditions, Murashige and Skoog medium supplemented with indole-3-butyric acid resulted in the greatest adventitious root induction rate. The amount of the major active component of the adventitious roots of Ama1, calycosin-7-O--D-glucopyranoside was higher than that of other adventitious root samples. Conclusions : These results suggest that the adventitious roots of A. membranaceus could be used for the commercial production of medicines.