Seung-Eun Oh

Discrimination of three Scrophularia plants utilizing 'Scrophularia Radix' by DNA markers based on internal transcribed spacer (ITS) sequences

One of the medicinal materials produced by plants belonging to the genus Scrophularia, ‘Scrophularia Radix’ (SR), has been prescribed for many centuries to treat diseases such as inflammation, abscesses of carbuncles and constipation. In China, the dried root of S. ningoensis Hemsley is the source of SR. In contrast, the root of S. buergeriana is generally prescribed as SR in Korea. Studies conducted to identify the bioactive compounds in these two Scrophularia plants have revealed marked differences in the contents and concentration of the compounds they contain. However, S. ningpoensis has been indiscriminately prescribed in Korea along with S. buergeriana as SR. Furthermore, S. koraiensis has long been used in lieu of S. buergeriana as SR in Korea. Therefore, a standard or method to reliably distinguish these three species of Scrophularia is needed. Recently, we found that the differences in the nucleotide sequences of the internal transcribed spacer (ITS) of Scrophularia plants could be applied to develop DNA markers to discriminate each plant. In this study, ITS sequences of 22 samples including three types of Scrophularia plants were amplified, determined and analyzed. Based on the results of these analyses, we designed the following primer sets: Ni F (5′-TTAACCATATAGGGGCCTCG-3′) / Ni R (5′-C CCCTCTCTGTATCCCAA-3′) to amplify a 379 bp DNA marker for the identification of S. ningpoensis; Bu F (5′-TTAACC ATATCGGGGCCAAG-3′) / Bu R (5′-ATCACGACAGCAC GCGA-3′) to amplify a 491 bp DNA marker for S. buergeriana; and Ko F (5′-ATAACCATATCGGGGCCTC-3′) / Ko R (5′-TCAAGAAACGCACTATCCC-3′) to amplify a 167 bp DNA marker of S. koraiensis. Using these primer sets, we were able to efficiently identify Scrophularia plants sold as SR in the herbal market in dried and sliced states after processing as medicinal materials.

The inrolling phenomena of petals during senescence in cut carnations (Dianthus caryophyllus L cv. shinkibo)

The inward rolling of the petals is one of typical symptoms observed in the process of climacteric corolla senescence. Inrolling was mimicked by treating the lower part of the petal instead of the whole petal of cut carnations (cv. Shinkibo) with exogenous ethylene. In these petal segments, the climacteric ethylene burst occurred right at the inrolling stage, indicating that these petal segments may be an excellent model system for examining corolla senescence. According to kinetic analysis, an asymmetry in the lengths of the adaxial and abaxial sides of petal segments appeared to be the direct cause of the inward rolling. While the length of the abaxial side of the transverse section of petal segments increased during the analysis, the ultimate length of the adaxial side was shrunken by the same ethylene action. Interestingly, the kinetics curve of the adaxial side consisted of two distinct phases. The rate of expansion/shrink of either side of the petal and the slope of each phase varied with the chemicals affected in the rolling process of the petal segments: e.g., n-octanoic acid, polyamines, and inhibitors of the Ca2+-channel blocker.

Developmental Ultrastructure of Glandular Trichomes of Rosmarinus officinalis: Secretory Cavity and Secretory Vesicle Formation

Glandular trichomes in the leaf lamina of Rosmarinus officinalis L. were examined by scanning and transmission electron microscopy. The leaves were characterized by an abundance of two types of glandular trichomes—small capitate and large peltate glandular trichomes. In addition to the glandular trichomes, numerous non-glandular trichomes were present on the abaxial surface of the leaf. These trichomes mainly predominated on the midrib, whereas glandular trichomes occurred on non-vein areas. At the initial phase of secretory cavity formation, hyaline areas were abundant in periclinal walls of head cells, while they were not observed in the anticlinal walls. The hyaline areas gradually increased in size, fusing with other areas throughout the wall. Loose wall material adjacent to hyaline areas was released from the head cell walls and migrated into the secretory cavities. As the secretory cavities continued to enlarge, the new vesicles emerging into the secretory cavities from the walls of head cells became surrounded with the surface of a typical membrane. They developed a round shape, but the contours of the vesicle surfaces appeared polygonal when tightly packed inside a cavity. These vesicles varied in size; small vesicles often possessed electron-dense contents, while large vesicles contained electron-light contents.

Application of Partial Internal Transcribed Spacer Sequences for the Discrimination of Artemisia capillaris from Other Artemisia Species

Several Artemisia species are used as herbal medicines including the dried aerial parts of Artemisia capillaris, which are used as Artemisiae Capillaris Herba (known as “Injinho” in Korean medicinal terminology and “Yin Chen Hao” in Chinese). In this study, we developed tools for distinguishing between A. capillaris and 11 other Artemisia species that grow and/or are cultured in China, Japan, and Korea. Based on partial nucleotide sequences in the internal transcribed spacer (ITS) that differ between the species, we designed primers to amplify a DNA marker for A. capillaris. In addition, to detect other Artemisia species that are contaminants of A. capillaris, we designed primers to amplify DNA markers of A. japonica, A. annua, A. apiacea, and A. anomala. Moreover, based on random amplified polymorphic DNA analysis, we confirmed that primers developed in a previous study could be used to identify Artemisia species that are sources of Artemisiae Argyi Folium and Artemisiae Iwayomogii Herba. By using these primers, we found that multiplex polymerase chain reaction (PCR) was a reliable tool to distinguish between A. capillaris and other Artemisia species and to identify other Artemisia species as contaminants of A. capillaris in a single PCR.

Differentiating Coptis chinensis from Coptis japonica and other Coptis species used in Coptidis Rhizoma based on partial trnL-F intergenic spacer sequences

Dried rhizomes of Coptis species are utilized as “Coptidis Rhizoma” (CR), an important herbal medicinal material in traditional Chinese medicine. Almost all CRs traded in the Korean herbal medicine market originate from Coptis chinensis (“Chun Hwang-Lyun” in Korean medical terminology). Other minor CRs originate from Coptis japonica (“Il Hwang-Lyun”). Although there is an obvious discrepancy in the price of traded CRs in the herbal market depending on the Coptis species, CRs originating from C. chinensis and C. japonica are often confused. Furthermore, the CR traded as “Chun Hwang-Lyun” is occasionally mixed with rhizomes of Coptis deltoidea and/or Coptis omeiensis. Therefore, we sought to discriminate C. chinensis from C. japonica, as well as C. deltoidea and C. omeiensis, by using nucleotide sequence differences in the partial trnL-F intergenic spacer. We developed an efficient real-time polymerase chain reaction (PCR)-based discrimination assay to separate samples of C. chinensis from those of C. japonica without the need to separate the DNA markers by using gel electrophoresis. In addition, we developed a multiplex PCR method with which we were able to discriminate samples of C. chinensis from those of C. deltoidea and C. omeiensis by amplifying the 153-bp DNA marker in C. chinensis in a single PCR process.

A PCR-based assay for discriminating Cervus and Rangifer (Cervidae) antlers with mitochondrial DNA polymorphisms1

This study describes a method for discriminating Rangifer antlers from true Cervus antlers using agarose gel electrophoresis, capillary electrophoresis, quantitative real-time PCR, and allelic discrimination. Specific primers labeled with fluorescent tags were designed to amplify fragments from the mitochondrial D-loop genes for various Cervus subspecies and Rangifer tarandus differentially. A 466-bp fragment that was observed for both Cervus and Rangifer antlers served as a positive control, while a 270-bp fragment was specifically amplified only from Rangifer antlers. Allelic discrimination was used to differentiate between Cervus and Rangifer antlers, based on the amplification of specific alleles for both types of antlers. These PCR-based assays can be used for forensic and quantitative analyses of Cervus and Rangifer antlers in a single step, without having to obtain any sequence information. In addition, multiple PCR-based assays are more accurate and reproducible than a single assay for species-specific analysis and are especially useful in this study for the identification of original Cervus deer products from fraudulent Rangifer antlers.

Suppression of Ca2+-Influx after ACC-uptake by spermine inVigna radiata

Exogenous ACC-induced ethylene production in mung bean hypocotyl segments was severely inhibited by the treatment of spermine in a 6 hr incubation. In protoplasts acquired from the same tissues and pretreated with ACC, the concentration of cytosolic Ca2+ was increased after the addition of exogenous Ca2+. However, this increase of Ca2+ concentration was strongly suppressed by spermine. In previous studies, an artifical increase of Ca2+ influx by the treatment of a Ca2+-ionophore stimulated ACC-induced ethylene production. The inhibitory effect of spermine on ACC-induced ethylene production was more prominent than that of putrescine which has fewer NH3+ groups than spermine. In addition, spermine more prominently suppressed ACC-induced ethylene production in protoplasts in which fewer Ca2+ ions were released from Ca2+-storage organelles. Also, the amount of transcript of ACC-oxidase which converts ACC to ethylene was decreased by the treatment of spermine. However, this reduction resulted only through the suppression of ethylene production in a 2 hr incubation of mung bean segments. On the basis of these results, we suggest that there is a coupling of ACC-uptake to the increase of cytosolic Ca2+ concentration. In addition, the reduction of exogenous ACC-induced ethylene production by spermine could have resulted, at least partially, from reducing the Ca2+ influx which stimulates ACC-oxidase activity.

Root Gravitropic Response of Phytochrome Mutant ( phyAB) in Arabidopsis

Phytochrome double mutant (phyAB) showed the delayed root gravitropic response compared to the wild type (WT) in Arabidopsis. After 8 hr of gravistimulation, the gravitropic response of mutant showed 48% of the WT. The delayed response started at 1.5 hr after gravistimulation. And we measured the ethylene production in the root segments of WT and mutant for 12 hr. Ethylene production of mutant decreased about 40% of the WT at 12 hr. This result suggested that the phytochrome might be linked with ethylene production in some way. Generally, ethylene inhibits the growth of plant organs including roots. We measured the root growth rate in the presence of ACC (1-aminocyclopropane-1-carboxylic acid), a precursor of ethylene. And WT showed the inhibition of root growth with ACC, but mutant did not show the inhibition as WT did. To confirm the relationship between the ethylene and gravitropic response, we measured the gravitropic response with ACC. In the presence of 10-6 M ACC, WT showed the 37.4% inhibition compared to the control (no ACC), whereas mutant showed the only 6.6% inhibition of control (no ACC). This research suggested the relationship between phytochrome and gravitropic response through an ethylene production.

Effect of 1-aminocyclopropane-1-carboxylic acid (ACC)-induced ethylene on cellulose synthase A (CesA) genes in flax (Linum usitatissimum L. ‘Nike’) seedlings

IntroductionCellulose microfibril is a major cell wall polymer that plays an important role in the growth and development of plants. The gene cellulose synthase A (CesA), encoding cellulose synthases, is involved in the synthesis of cellulose microfibrils. However, the regulatory mechanism of CesA gene expression is not well understood, especially during the early developmental stages.ObjectiveTo identify factor(s) that regulate the expression of CesA genes and ultimately control seedling growth and development.MethodsThe presence of cis-elements in the promoter region of the eight CesA genes identified in flax (Linum usitatissimum L. ‘Nike’) seedlings was verified, and three kinds of ethylene-responsive cis-elements were identified in the promoters. Therefore, the effect of ethylene on the expression of four selected CesA genes classified into Clades 1 and 6 after treatment with 10−4 and 10−3 M 1-aminocyclopropane-1-carboxylic acid (ACC) was examined in the hypocotyl of 4–6-day-old flax seedlings.ResultsACC-induced ethylene either up- or down-regulated the expression of the CesA genes depending on the clade to which these genes belonged, age of seedlings, part of the hypocotyl, and concentration of ACC.ConclusionEthylene might be one of the factors regulating the expression of CesA genes in flax seedlings.

A PCR-based assay for discriminating and (Cervidae) antlers with mitochondrial DNA polymorphisms

This study describes a method for discriminating Rangifer antlers from true Cervus antlers using agarose gel electrophoresis, capillary electrophoresis, quantitative real-time PCR, and allelic discrimination. Specific primers labeled with fluorescent tags were designed to amplify fragments from the mitochondrial D-loop genes for various Cervus subspecies and Rangifer tarandus differentially. A 466-bp fragment that was observed for both Cervus and Rangifer antlers served as a positive control, while a 270-bp fragment was specifically amplified only from Rangifer antlers. Allelic discrimination was used to differentiate between Cervus and Rangifer antlers, based on the amplification of specific alleles for both types of antlers. These PCR-based assays can be used for forensic and quantitative analyses of Cervus and Rangifer antlers in a single step, without having to obtain any sequence information. In addition, multiple PCR-based assays are more accurate and reproducible than a single assay for species-specific analysis and are especially useful in this study for the identification of original Cervus deer products from fraudulent Rangifer antlers.