Kyung Hwan Boo

Distribution and Biosynthesis of 20-Hydroxyecdysone in Plants of Achyranthes japonica Nakai

There is increasing interest in phytoecdysteroids (PEs) because of their potential role in plant defense against insects. To understand the mechanism regulating their levels in plants, the fluctuation, distribution, and biosynthesis of PE 20-hydroxyecdysone (20E) examined in Achyranthes japonica. The total amount of 20E per individual plant initially remained at a constant level, and increased markedly after the first leaf pair (LP) stage, while the concentration of 20E in a given plant decreased rapidly during vegetative growth. In addition, the incorporation of [2-14C]-mevalonic acid into 20E did not differ significantly depending on plant organs and developmental stages, suggesting that biosynthesis of 20E is not restricted to particular organs or growth stages.

Evaluation of antioxidant potential of ethyl acetate fraction of Rosmarinus officinalis L. and its major components

Abstract The solvent fractions of rosemary methanol extract were obtained by successive extraction with n-hexane, chloroform, ethyl acetate, butanol, and water. The ethyl acetate fraction (EAF) contained a remarkable amount of polyphenol and flavonoid as well as high levels of alkyl and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging activity. The activity guided fractionation and repeated chromatographic separations over silica gel, RP C18, and Sephadex LH-20 led to isolation of six compounds from the EAF. 1H NMR, 13C NMR, 2D NMR, MS, and IR spectroscopies determined the compounds to be caffeic acid (1), rosmarinic acid (2), rosmarinic acid methyl ester (3), luteolin (4), apigenin (5), and hispidulin (6), and high-performance liquid chromatography quantification was used to determine concentrations in EAF. Among the six isolated compounds, rosmarinic acid methyl ester showed the highest scavenging activities against di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium, alkyl and ABTS radicals. The EAF mixture, but not individual isolated compounds, shielded dermal fibroblast cells from H2O2-induced cytotoxicity at concentrations that encompass the SC50 of alkyl and ABTS radical. Therefore, our findings suggested for the first time that antioxidant capacity of the EAF mixture result in a synergistic effect on the antioxidant action.

Construction and profiling of a cDNA library from young fruit of satsuma mandarin

To profile gene expression in the early stage of fruit development from ‘Nichinan No. 1’ satsuma mandarin (Citrus unshiu Marc.), we isolated total mRNA at 30 d after flowering. A cDNA library was prepared from mature mRNAs and a total of 2350 cDNA clones were partially sequenced. In all, 1914 ESTs were acquired after the removal of the vector sequence and filtering over a minimum length of 150 nucleotides. A total of 763 unigenes, consisting of 138 contigs and 625 singletons, was identified after assembly of those ESTs. According to our homology search with BLASTX against the NCBI database, the deduced amino acid sequences of 253 unigenes were homologous to proteins with known function and 242 unigenes were significantly matched to proteins with putative or unknown functions. The remaining 268 showed no significant similarity to any protein sequences found in the public database with matches higher than an E value of 10-5. The 253 unigenes matched to proteins with known function were then manually assigned to 10 cellular functional categories using a modified MIPS MATDB classification. The expression level of each gene was analyzed based on the redundancy of cDNA clones in each contig that comprised more than 10 ESTs. Here, the most abundant gene expressed in young fruits was for a chitinase precursor. A miraculin-like protein and a lectin-related protein precursor were also abundant.

In vitro plant regeneration of Aster scaber via somatic embryogenesis

We established an in vitro plant regeneration system via somatic embryogenesis of Aster scaber, an important source of various biologically active phytochemicals. We examined the callus induction and embryogenic capacities of three explants, including leaves, petioles, and roots, on 25 different media containing different combinations of α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). The optimum concentrations of NAA and BA for the production of embryogenic calli were 5.0 μM and 0.05 μM, respectively. Media containing higher concentrations of auxin and cytokinin (such as 25 μM NAA and 25 μM BA) were suitable for shoot regeneration, especially for leaf-derived calli, which are the most readily available calli and are highly competent. For root induction from regenerated shoots, supplemental auxin and/or cytokinin did not improve rooting, but instead caused unwanted callus induction or retarded growth of regenerated plants. Therefore, plant growth regulator-free medium was preferable for root induction. Normal plants were successfully obtained from calli under the optimized conditions described above. This is the first report of the complete process of in vitro plant regeneration of A. scaber via somatic embryogenesis. Graphical Abstract Plant regeneration of A. scaber via somatic embryogenesis

Fluctuation of 20-hydroxyecdysone in Individual Organs of Achyranthes japonica during Reproductive Growth Stage and Its Accumulation into Seed

To better understand 20-hydroxyecdysone (20E) fluctuation and accumulation in perennial plant, 20E concentration in individual organs of Achyranthes japonica during reproductive growth stage were analyzed by high performance liquid chromatography (HPLC). Concentrations of 20E in root and floral part were much higher than those in stem and leaf during reproductive growth stage and rapidly increased from flowering stage in August to seed-setting stage in October, and thereafter decreased at the stage of seed maturing in November. In contrast, the 20E concentrations in stem and leaf gradually decreased during reproductive growth. In the analysis of detailed fluctuation of 20E in floral part, the 20E concentration was highest in the seed at the early stage of seed development, compared to flower, peduncle, seed coat, and/or seed in other growth stages, and decreased during seed maturation. The accumulation of 20E in reproductive organs, especially seed and root, suggests that 20E has a defensive role for protection of especially newly developing organs against phytophagous insects.

Anti-viral activity of blue chanterelle (Polyozellus multiplex) that inhibits α-glucosidase

Blue chanterelle (Polyozellus multiplex), known as an edible mushroom, was extracted using methanol to screen on anti-viral agent. Syncytium formation in Newcastle disease virus (NDV)-infected baby hamster kidney (BHK) cell originates from the trafficking of viral glycoprotein into cell-surface. Blue chanterelle inhibited not only syncytium formation, but also trafficking of glycoprotein, hemagglutinin-neuramidase (HN), onto cell-surface. Viral glycoprotein is processed within the endoplasmic reticulum during routing to surface. Blue chanterelle extracts showed the inhibitory activities (IC50 10 μg/mL) against α-glucosidase. These results suggested that blue chanterelle extracts inhibited the cell-surface expression of NDV-HN glycoprotein without significantly affecting HN glycoprotein synthesis in NDV-infected BHK cells.

The Antiviral Effects of Areca catechu L. Extract

Trafficking of viral glycoproteins to the cell surface results in syncytium formation in baby hamster kidney (BHK) cells infected with Newcastle disease virus (NDV). An extract from the medicinal Areca catechu L plant inhibited not only syncytium formation, but also trafficking of the hemagglutinin-neuramidase (HN) glycoprotein to the cell-surface. The viral glycoprotein was processed within the endoplasmic reticulum during transit to the cell membrane. Fungal extracts showed inhibitory activities (IC 50 10 µg/mL) against α-glucosidase. These results suggested that A. catechu L. extracts inhibited the cell-surface expression of NDV-HN glycoprotein without significantly affecting HN glycoprotein synthesis in NDV-infected BHK cells.

HIGLE is a bifunctional homing endonuclease that directly interacts with HYL1 and SERRATE in Arabidopsis thaliana

A highly coordinated complex known as the microprocessor precisely processes primary transcripts of MIRNA genes into mature miRNAs. In plants, the microprocessor minimally consists of three components: Dicer‐like protein 1 (DCL1), HYPONASTIC LEAF 1 (HYL1), and SERRATE (SE). To precisely modulate miRNA maturation, the microprocessor cooperates with at least 12 proteins in plants. In addition, we here show the involvement of a novel gene, HYL1‐interacting GIY‐YIG‐like endonuclease (HIGLE). The encoded protein has a GIY‐YIG domain that is generally found within a class of homing endonucleases. HIGLE directly interacts with the microprocessor components HYL1 and SE. Unlike the functions of other GIY‐YIG endonucleases, the catalytic core of HIGLE has both DNase and RNase activities that sufficiently processes miRNA precursors into short fragments in vitro.

A Profile of Expressed Sequence Tags in Newly Developing Leaves of Aralia elata Seem

A cDNA library was constructed from the newly developed young leaves of Aralia elata Seem, and a total of 2,755 cDNAs were partially sequenced. Sequences with high quality greater than 150 bp after trimming vector and ambiguous sequences resulted in 2,689 ESTs. These ESTs were clustered into 2,010 unigenes consisting of 376 contigs with two or more ESTs and 1,634 singletons. A homology search with BLASTX against the NCBI database identified 1,256 (63%) unigenes homologous to proteins of known or putative function, 350 (17%) genes assumed as proteins with unknown function, and 404 (20%) unigenes with no significant match to any protein sequence, including sequences with matches greater than the E value of −105 in the NCBI database. The expression level of each gene was analyzed based on the number of cDNA clones in each contig composed of at least 10 ESTs. The most abundant gene identified was acid phosphatase. The genes related to chlorophyll a-b binding protein, elongation factor 1-alpha, ribulose 1,5-bisphosphate carboxylase small subunit and cyclophilin were also plentiful. The 1,259 unigenes matched with proteins of known or putative function were classified into a functional category using the MIPS BLAST program and 616 unigenes were assigned to putative biochemical functions. Furthermore, five, sixteen and twelve unigenes in our EST set were assumed as proteins involved in squalene synthesis, cytochrome P450 and glycosyltransferase, respectively. These genes should be further explored for their involvement in the saponin biosynthesis pathway in A. elata Seem.