Yi Lee

Postembryonic Seedling Lethality in the Sterol-Deficient Arabidopsis cyp51A2 Mutant Is Partially Mediated by the Composite Action of Ethylene and Reactive Oxygen Species

Seedling-lethal phenotypes of Arabidopsis (Arabidopsis thaliana) mutants that are defective in early steps in the sterol biosynthetic pathway are not rescued by the exogenous application of brassinosteroids. The detailed molecular and physiological mechanisms of seedling lethality have yet to be understood. Thus, to elucidate the underlying mechanism of lethality, we analyzed transcriptome and proteome profiles of the cyp51A2 mutant that is defective in sterol 14α-demethylation. Results revealed that the expression levels of genes involved in ethylene biosynthesis/signaling and detoxification of reactive oxygen species (ROS) increased in the mutant compared with the wild type and, thereby, that the endogenous ethylene level also increased in the mutant. Consistently, the seedling-lethal phenotype of the cyp51A2 mutant was partly attenuated by the inhibition of ethylene biosynthesis or signaling. However, photosynthesis-related genes including Rubisco large subunit, chlorophyll a/b-binding protein, and components of photosystems were transcriptionally and/or translationally down-regulated in the mutant, accompanied by the transformation of chloroplasts into gerontoplasts and a reduction in both chlorophyll contents and photosynthetic activity. These characteristics observed in the cyp51A2 mutant resemble those of leaf senescence. Nitroblue tetrazolium staining data revealed that the mutant was under oxidative stress due to the accumulation of ROS, a key factor controlling both programmed cell death and ethylene production. Our results suggest that changes in membrane sterol contents and composition in the cyp51A2 mutant trigger the generation of ROS and ethylene and eventually induce premature seedling senescence.

Psychrotolerant Endophytic Pseudomonas sp. Strains OB155 and OS261 Induced Chilling Resistance in Tomato Plants (Solanum lycopersicum Mill.) by Activation of Their Antioxidant Capacity.

Studies on chilling stress damage and its mitigation through microorganisms in members of family Solanaceae is limited, despite their economic importance. We studied chilling stress alleviation in tomato plants colonized by psychrotolerant bacterial strains Pseudomonas vancouverensis OB155-gfp and P. frederiksbergensis OS261-gfp. Log phase cultures of bacterial strains were coated on surface-sterilized seeds (bacterization) before sowing and nonbacterized (control) seeds were coated with sterile bacterial growth medium. All plants were grown at temperatures of 30 and 25°C and at the end of 4 weeks, chilling treatment (12 and 10°C) was imposed for 1 week on half of the bacterized and control plants. Under normal conditions (30 and 25°C), no significant difference was observed in antioxidant activity, proline accumulation, and expression of cold acclimation genes in tomato leaf tissues of both control and bacterized plants. However, plants exposed to temperatures of 12 and 10°C were found to decrease in robustness and nutrient uptake, accompanied by increased membrane damage. Chilling resistance in bacterized plants was evident from reduced membrane damage and reactive oxygen species levels, improved antioxidant activity in leaf tissues, and high expression of cold acclimation genes LeCBF1 and LeCBF3 compared with control plants. Confocal microscopy confirmed effective colonization and intercellular localization of cold-adapted bacterial strains OB155-gfp and OS261-gfp.

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.

A Combination of Biochemical and Proteomic Analyses Reveals Bx-LEC-1 as an Antigenic Target for the Monoclonal Antibody 3-2A7-2H5-D9-F10 Specific to the Pine Wood Nematode

Pine wilt disease (PWD) is one of the most devastating forest diseases in Asia and Europe. The pine wood nematode, Bursaphelenchus xylophilus, has been identified as the pathogen underlying PWD, although the pathology is not completely understood. At present, diagnosis and confirmation of PWD are time consuming tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for B. xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically, but not those from other closely related nematodes. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions, or bands isolated from SDS-PAGE, two-dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two nonidentical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to d-(+)-raffinose and N-acetyllactosamine were much higher than that to l-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.

Genome and evolution of the shade‐requiring medicinal herb Panax ginseng

Summary Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane‐type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome‐scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics‐assisted breeding or metabolic engineering.

Genetic diversity analysis of Glycyrrhiza uralensis using 8 novel polymorphic microsatellite markers

Licorice plant (Glycyrrhiza spp.) is an important herb, but the major portion of the national demand is imported to Korea because the domestic production base is vulnerable. We performed basic molecular breeding research for domestic cultivation and production. All publicly available G. uralensis EST sequences, which totaled 56,089, were assembled into 4,821 unigenes and examined for microsatellites. Eight polymorphic microsatellite loci were identified and 16 G. uralensis and 6 G. glabra accessions, which were collected from different locations, were genotyped using the microsatellites. Genetic diversity within the accessions was estimated by construction of a dendrogram. The dendrogram was clustered into two groups. The results showed that there is a correlative genetic relationship between species. The microsatellite markers were found to be useful for diversity analysis as they are able to successfully distinguish the Glycyrrhiza accessions.

Comprehensive analysis of AHL homologous genes encoding AT-hook motif nuclear Localized protein in rice

The AT-hook motif is a small DNA-binding protein motif that has been found in the high mobility group of non-histone chromosomal proteins. The Arabidopsis genome contains 29 genes encoding the AT-hook motif DNA-binding protein (AHL). Recent studies of Arabidopsis genes (AtAHLs) have revealed that they might play diverse functional roles during plant growth and development. In this report, we mined 20 AHL genes (OsAHLs) from the rice genome database using AtAHL genes as queries and characterized their molecular features. A phylogenetic tree revealed that OsAHL proteins can be classified into 2 evolutionary clades. Tissue expression pattern analysis revealed that all of the OsAHL genes might be functionally expressed genes with 3 distinct expression patterns. Nuclear localization analysis using transgenic Arabidopsis showed that several OsAHL proteins are exclusively localized in the nucleus, indicating that they may act as architectural transcription factors to regulate expression of their target genes during plant growth and development.

Development of 44 Novel Polymorphic SSR Markers for Determination of Shiitake Mushroom (Lentinula edodes) Cultivars

The shiitake mushroom (Lentinula edodes) is one of the most popular edible mushrooms in the world and has attracted attention for its value in medicinal and pharmacological uses. With recent advanced research and techniques, the agricultural cultivation of the shiitake mushroom has been greatly increased, especially in East Asia. Additionally, demand for the development of new cultivars with good agricultural traits has been greatly enhanced, but the development processes are complicated and more challenging than for other edible mushrooms. In this study, we developed 44 novel polymorphic simple sequence repeat (SSR) markers for the determination of shiitake mushroom cultivars based on a whole genome sequencing database of L. edodes. These markers were found to be polymorphic and reliable when screened in 23 shiitake mushroom cultivars. For the 44 SSR markers developed in this study, the major allele frequency ranged from 0.13 to 0.94; the number of genotypes and number of alleles were each 2–11; the observed and expected heterozygosity were 0.00–1.00 and 0.10–0.90, respectively; and the polymorphic information content value ranged from 0.10 to 0.89. These new markers can be used for molecular breeding, the determination of cultivars, and other applications.

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.

Simple sequence repeat marker development from Codonopsis lanceolata and genetic relation analysis

In this study, we developed 15 novel polymorphic simple sequence repeat (SSR) markers by SSR-enriched genomic library construction from Codonopsis lanceolata. We obtained a total of 226 non-redundant contig sequences from the assembly process and designed primer sets. These markers were applied to 53 accessions representing the cultivated C. lanceolata in South Korea. Fifteen markers were sufficiently polymorphic, and were used to analyze the genetic relationships between the cultivated C. lanceolata. One hundred three alleles of the 15 SSR markers ranged from 3 to 19 alleles at each locus, with an average of 6.87. By cluster analysis, we detected clear genetic differences in most of the accessions, with genetic distance varying from 0.73 to 0.93. Phylogenic analysis indicated that the accessions that were collected from the same area were distributed evenly in the phylogenetic tree. These results indicate that there is no correlative genetic relationship between geographic areas. These markers will be useful in differentiating C. lanceolata genetic resources and in selecting suitable lines for a systemic breeding program.