Shin-Woo Lee

YUCCA6 over-expression demonstrates auxin function in delaying leaf senescence in Arabidopsis thaliana

The Arabidopsis thaliana YUCCA family of flavin monooxygenase proteins catalyses a rate-limiting step in de novo auxin biosynthesis. A YUCCA6 activation mutant, yuc6-1D, has been shown to contain an elevated free IAA level and to display typical high-auxin phenotypes. It is reported here that Arabidopsis plants over-expressing YUCCA6, such as the yuc6-1D activation mutant and 35S:YUC6 transgenic plants, displayed dramatic longevity. In addition, plants over-expressing YUCCA6 exhibited classical, delayed dark-induced and hormone-induced senescence in assays using detached rosette leaves. However, plants over-expressing an allele of YUCCA6, that carries mutations in the NADPH cofactor binding site, exhibited neither delayed leaf senescence phenotypes nor phenotypes typical of auxin overproduction. When the level of free IAA was reduced in yuc6-1D by conjugation to lysine, yuc6-1D leaves senesced at a rate similar to the wild-type leaves. Dark-induced senescence in detached leaves was accompanied by a decrease in their free IAA content, by the reduced expression of auxin biosynthesis enzymes such as YUCCA1 and YUCCA6 that increase cellular free IAA levels, and by the increased expression of auxin-conjugating enzymes encoded by the GH3 genes that reduce the cellular free auxin levels. Reduced transcript abundances of SAG12, NAC1, and NAC6 during senescence in yuc6-1D compared with the wild type suggested that auxin delays senescence by directly or indirectly regulating the expression of senescence-associated genes.

Overexpression of Arabidopsis YUCCA6 in Potato Results in High-Auxin Developmental Phenotypes and Enhanced Resistance to Water Deficit

Indole-3-acetic acid (IAA), a major plant auxin, is produced in both tryptophan-dependent and tryptophan-independent pathways. A major pathway in Arabidopsis thaliana generates IAA in two reactions from tryptophan. Step one converts tryptophan to indole-3-pyruvic acid (IPA) by tryptophan aminotransferases followed by a rate-limiting step converting IPA to IAA catalyzed by YUCCA proteins. We identified eight putative StYUC (Solanum tuberosum YUCCA) genes whose deduced amino acid sequences share 50%-70% identity with those of Arabidopsis YUCCA proteins. All include canonical, conserved YUCCA sequences: FATGY motif, FMO signature sequence, and FAD-binding and NADP-binding sequences. In addition, five genes were found with ~50% amino acid sequence identity to Arabidopsis tryptophan aminotransferases. Transgenic potato (Solanum tuberosum cv. Jowon) constitutively overexpressing Arabidopsis AtYUC6 displayed high-auxin phenotypes such as narrow downward-curled leaves, increased height, erect stature, and longevity. Transgenic potato plants overexpressing AtYUC6 showed enhanced drought tolerance based on reduced water loss. The phenotype was correlated with reduced levels of reactive oxygen species in leaves. The results suggest a functional YUCCA pathway of auxin biosynthesis in potato that may be exploited to alter plant responses to the environment.

Expressional characterization of dehydroascorbate reductase cDNA in transgenic potato plants

In plants ascorbic acid (AsA) is a strong antioxidant or reductant that can be converted to dehydroascorbate (DHA) by oxidation. DHA, a very short-lived chemical, can either be hydrolyzed irreversibly to 2,3-diketogulonic acid or recycled to AsA by dehydroascorbate reductase (DHAR).DHAR cDNA, isolated from sesame hairy roots, was inserted into two plant expression vector syrtems with theCaMV35S promoter (CaMV35S : :DHAR) and a potato tuber-specific promoter,Patatin (Patatin : :DHAR). Southern and northern blot hybridization analyses indicated thatDHAR cDNA was successfully integrated into the potato genome and actively transcribed. High levels of sesameDHAR transcript and DHAR enzyme activity were determined, by thePatatin promoter, in regenerated potato tubers, but their levels in leaves were very low. In contrast, much higher amounts of transcript were accumulated in the leaves of CaMV35S : :DHAR regenerants than in the tubers while the activity of DHAR enzyme was higher in the latter. AsA content in the tubers of Patatin : :DHAR transgenic lines was also increased (1.1- to 1.3-fold) compared with that of non-transgenic plants. However, this was not true for the transgenic leaves. In contrast, theCaMV35S promoter was associated with AsA accumulations in both the tubers (up to 1.6-fold) and the leaves (up to 1.5-fold). However, more detailed analyses indicated that this increased enzyme activity was not always accompanied by an elevation in AsA content from transgenic plants. This suggests that other factors may limit the accumulation of vitamin C via ascorbate-recycling in transgenic potato plants.

Overexpression of the sweet potato IbOr gene results in the increased accumulation of carotenoid and confers tolerance to environmental stresses in transgenic potato.

In a previous study, we have evidenced that the overexpression of the IbOr gene isolated from sweet potato conferred a tolerance activity against salinity and methyl viologen (MV) treatment in transgenic sweet potato calli along with an enhanced carotenoid content. In this study, to further examine the function of the IbOr gene in heterologous organism, we transformed the IbOr gene into potato under the direction of SWPA2 promoter, a strong inducible promoter upon treatment with various environmental stresses. Consistently with our previous study of sweet potato calli, the level of total carotenoid was elevated up to 2.7-fold (38.1 μg g(-1)DW) compared to the non-transgenic control, Atlantic cultivar. However, the composition of carotenoid was not influenced by the overexpression of the IbOr gene since only pre-existing carotenoids in the non-transgenic control including violaxanthin, lutien and β-carotene were elevated at a similar level of total carotenoids. In general, the transcript levels for most of carotenogenesis-related genes were elevated in transgenic tuber, whereas they remained at similar levels in transgenic leaf tissues compared to those of non-transgenic controls. The increased levels of carotenoid content in the leaf or tuber tissue of transgenic lines were correlated with the enhanced tolerance activity against salt- or MV-mediated oxidative stresses and DPPH radical-scavenging activity. Our preliminary results suggest that further investigation is required for the development of a crop tolerant to salinity and other environmental stresses through the overexpression of the IbOr gene.

Development of molecular markers, based on chloroplast and ribosomal DNA regions, to discriminate three popular medicinal plant species, Cynanchum wilfordii, Cynanchum auriculatum, and Polygonum multiflorum

Identification of plant species is important for standardizing herbal medicine. Cynanchum wilfordii (Baekshuoh in Korean) and Polygonum multiflorum (Hashuoh in Korean) are important oriental medicinal herbs in Korea, Japan, and China. Cynanchum auriculatum is a faster growing and more productive plant than C. wilfordii; and, it is not recognized as a medicinal plant in the Korean Pharmacopoeia. C. wilfordii, P. multiflorum, and C. auriculatum are often misidentified in the Korean herbal medicine marketplace due to their morphological similarities and similar names. In this study, we investigated molecular authentication of these three medicinal plants using DNA sequences in the TrnL-F chloroplast intergenic region. Specific species identification was achieved by detecting allelic variations of single nucleotide polymorphisms (SNPs) using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and high resolution melting curve analysis. Our results demonstrate that the intraspecific genetic distance between C. wilfordii and C. auriculatum is relatively low. We also developed a quantitative PCR assay using species-specific TrnL-F primers, which allowed us to estimate the ratio of C. wilfordii and C. auriculatum using varying ratios of mixed genomic DNA template from the two species. Additionally, to identify species in hybrid plants produced by cross-fertilization, we analyzed nuclear ribosomal DNA internal transcribed spacer regions in C. wilfordii and C. auriculatum by ARMS-PCR. Our results indicate that SNP-based molecular markers, usable to barcode tools could provide efficient and rapid authentication of these closely related medicinal plant species, and will be useful for preventing the distribution of products contaminated with adulterants.

The sweetpotato ADP-glucose pyrophosphorylase gene (ibAGP1) promoter confers high-level expression of the GUS reporter gene in the potato tuber

Molecular farming refers to the process of creating bioengineered plants with the capability of producing potentially valuable products, such as drugs, vaccines, and chemicals. We have investigated the potential of the sweet potato ADP-glucose pyrophosphorylase gene (ibAGP1) promoter and its transit peptide (TP) as an expression system for the mass production of foreign proteins in potato. The ibAGP1 promoter and its TP sequence were transformed into potato along with beta-glucuronidase (GUS) as a reporter gene, and GUS activity was subsequently analyzed in the transgenic potato plants. In tuber tissues, GUS activity in transgenic plants carrying only the ibAGP1 promoter (ibAGP1::GUS) increased up to 15.6-fold compared with that of transgenic plants carrying only the CaMV35S promoter (CaMV35S::GUS). GUS activity in transgenic plants was further enhanced by the addition of the sweetpotato TP to the recombinant vector (ibAGP1::TP::GUS), with tuber tissues showing a 26-fold increase in activity compared with that in the CaMV35S::GUS-transgenic lines. In leaf tissues, the levels of GUS activity found in ibAGP1::GUS-transgenic lines were similar to those in CaMV35S::GUS-lines, but they were significantly enhanced in ibAGP1::TP::GUS-lines. GUS activity gradually increased with increasing tuber diameter in ibAGP1::GUS-transgenic plants, reaching a maximum level when the tuber was 35 mm in diameter. In contrast, extremely elevated levels of GUS activity - up to about 10-fold higher than that found in CaMV35S::GUS-lines - were found in ibAGP1::TP::GUS-transgenic lines at a much earlier stage of tuber development (diameter 4 mm), and these higher levels were maintained throughout the entire tuber developmental stage. These results suggest that the sweetpotato ibAGP1 promoter and its TP are a potentially strong foreign gene expression system that can be used for molecular farming in potato plants.

Effect of Drying Methods on the Saponin and Mineral Contents of Platycodon grandiflorum Radix

This study was conducted to provide basic information about the drying methods (daylight, hot-air, and freeze drying) used for Platycodon grandiflorum radix. We investigated the mineral, free sugar, and saponin contents of dried P. grandiflorum. The potassium and calcium contents of hot-air-dried samples were the highest (22.6 and 9.2 mg%, respectively), when compared to those of daylightor freeze-dried samples. Glucose and sucrose contents were the highest in freeze-dried samples (1,552 and 145.0 mg%, respectively), while fructose content was the highest in hot-air-dried samples (611.9 mg%). Platycodin D content was the highest in hot-air-dried samples (622.0 mg%); however platycodin D3, polygalacin D, and deapioplatycodin D contents were the highest in daylight-dried plant (113.5, 756.6, and 109.2 mg%, respectively). Glucose content was highly negatively correlated (p<0.01) with platycodin D, platycodin D3, and deapioplatycodin D (−0.924, −0.957, −0.861, p<0.01, respectively). These results suggest that the drying method affects the saponin content of P. grandiflorum and daylight and hot-air drying methods are more suitable and beneficial than freeze-drying.

Quantitative Analysis of Carotenoids in Carrot Cultivars Produced in Korea

Carrots (Daucus carota L.) are consumed as an important dietary source of provitamin A including -carotene, -carotene and lutein. An HPLC method was applied to determine the content of the carotenoid composition in carrot cutivars cultivated in Korea. HPLC analyses were carried out with five carrot cultivars (Socheon-5-chon, Hongsim-5-chon, Myeongju-5-chon, Seonhongbom-5-chon and Betarich) sown at April, 2007 and six cultivars (Yeoreum-5-chon, Hanyeoreum-5-chon, Sinheukjeon-5-chon, Bibariheukjeon, Manina and Betarich) sown at August of the same year. In general, the former varieties are not used for the sowing at summer because of their bolting (growth of floral axis). The former and the latter carrots were harvested after 110 and 96 days from seeding, respectively, and the carotenoids were extracted with acetone after freeze-drying. The amount of -carotene () was similar to that of -carotene () for the carrot cultivars sown at spring, while the content () was about a half of -carotene content () for the latter cultivars. In addition, the average content of lutein () in the former cultivars was eight times higher than that in the latter cultivars (). Among the spring cultivation types, Socheon-5-chon and Myeongju-5-chon showed higher amount of -carotene and -carotene, while the higher amount was determined in Yeoreum-5-chon and Sinheukjeon-5-chon among the autumn cultivation types. Validation of the HPLC-DAD method showed good linearity ( > 0.997) of the three compounds analyzed in a wide concentration range (). The R.S.D. values for intra-day and inter-day precision were less than 19.2% and the mean recovery of each compound was 85.4~104.7%.

Expressing the sweet potato orange gene in transgenic potato improves drought tolerance and marketable tuber production

Potato (Solanum tuberosum L.) is generally considered to be sensitive to drought stress. Even short periods of water shortage can result in reduced tuber production and quality. We previously reported that transgenic potato plants expressing the sweet potato orange gene (IbOr) under the control of the stress-inducible SWPA2 promoter (referred to as SOR plants) showed increased tolerance to methyl viologen-mediated oxidative stress and high salinity, along with increased carotenoid contents. In this study, in an effort to improve the productivity and environmental stress tolerance of potato, we subjected transgenic potato plants expressing IbOr to water-deficient conditions in the greenhouse. The SOR plants exhibited increased tolerance to drought stress under greenhouse conditions. IbOr expression was associated with slightly negative phenotypes, including reduced tuber production. Controlling IbOr expression imparted the same degree of drought tolerance while ameliorating these negative phenotypic effects, leading to levels of tuber production similar to or better than those of wild-type plants under drought stress conditions. In particular, under drought stress, drought tolerance and the production of marketable tubers (over 80g) were improved in transgenic plants compared with non-transgenic plants. These results suggest that expressing the IbOr transgene can lead to significant gains in drought tolerance and tuber production in potato, thereby improving these agronomically important traits.

Comparison of Isoflavone Contents and Antioxidant Effect in Cheonggukjang with Black Soybean Cultivars by Bacillus subtilis CSY191

BACKGROUND: Soybeans are the rich sources of isoflavones. To date, the changes of isoflavone contents in various black soybeans cheonggukjang during fermentation by Bacillus subtilis CSY191 has not been investigated.METHODS AND RESULTS: This study investigated the changes of total phenolic and isoflavone contents and antioxidant effects during cheonggukjang fermentation made with four black soybean (BS) cultivars including Cheongja, Cheongja#3, Geomjeong#5, and Ilpumgeomjeong with a potential probiotic Bacillus subtilis CSY191. The total phenolic contents, isoflavone-malonylglycoside and -aglycone contents, and antioxidant activity were increased in cheonggukjang at 48 h fermentation, while the content of isoflavone-glycosides was decreased during cheonggukjang fermentation. In particular, the Cheongja#3 soybean fermented at 37℃ for 48 h displayed the highest antioxidant activities, compared to those of the other BS cultivars tested. Also, the highest levels of total phenolic, daidzein, glycitein, and genistein were present at concentrations of 17.28 mg/g, 283.7 g/g, 39.9 g/g, and 13.2 g/g at the end of Cheongja#3 soybean fermentation.CONCLUSION: The results from this study suggested that the enhanced antioxidant activity of cheonggukjang of BS might be related to increased levels of total phenolic, isoflavon-aglycone, and malonyl-glycoside contents achieved during fermentation. Furthermore, fermented Cheongja#3 soybean showed the highest levels of enhanced antioxidant activities than the other BS cultivars.