Kang-Jin Cho

Genes up-regulated during red coloration in UV-B irradiated lettuce leaves

Molecular analysis of gene expression differences between green and red lettuce leaves was performed using the SSH method. BlastX comparisons of subtractive expressed sequence tags (ESTs) indicated that 7.6% of clones encoded enzymes involved in secondary metabolism. Such clones had a particularly high abundance of flavonoid-metabolism proteins (6.5%). Following SSH, 566 clones were rescreened for differential gene expression using dot-blot hybridization. Of these, 53 were found to overexpressed during red coloration. The up-regulated expression of six genes was confirmed by Northern blot analyses. The expression of chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), and dihydroflavonol 4-reductase (DFR) genes showed a positive correlation with anthocyanin accumulation in UV-B-irradiated lettuce leaves; flavonoid 3′,5′-hydroxylase (F3′,5′H) and anthocyanidin synthase (ANS) were expressed continuously in both samples. These results indicated that the genes CHS, F3H, and DFR coincided with increases in anthocyanin accumulation during the red coloration of lettuce leaves. This study show a relationship between red coloration and the expression of up-regulated genes in lettuce. The subtractive cDNA library and EST database described in this study represent a valuable resource for further research for secondary metabolism in the vegetable crops.

Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (Lactuca sativa)

The MYB transcription factors play important roles in the regulation of many secondary metabolites at the transcriptional level. We evaluated the possible roles of the Arabidopsis R2R3-MYB transcription factors in flavonoid biosynthesis because they are induced by UV-B irradiation but their associated phenotypes are largely unexplored. We isolated their genes by RACE-PCR, and performed transgenic approach and metabolite analyses in lettuce (Lactuca sativa). We found that one member of this protein family, AtMYB60, inhibits anthocyanin biosynthesis in the lettuce plant. Wild-type lettuce normally accumulates anthocyanin, predominantly cyanidin and traces of delphinidin, and develops a red pigmentation. However, the production and accumulation of anthocyanin pigments in AtMYB60-overexpressing lettuce was inhibited. Using RT-PCR analysis, we also identified the complete absence or reduction of dihydroflavonol 4-reductase (DFR) transcripts in AtMYB60- overexpressing lettuce (AtMYB60-117 and AtMYB60-112 lines). The correlation between the overexpression of AtMYB60 and the inhibition of anthocyanin accumulation suggests that the transcription factorAtMYB60 controls anthocyanin biosynthesis in the lettuce leaf. Clarification of the roles of the AtMYB60 transcription factor will facilitate further studies and provide genetic tools to better understand the regulation in plants of the genes controlled by the MYB-type transcription factors. Furthermore, the characterization of AtMYB60 has implications for the development of new varieties of lettuce and other commercially important plants with metabolic engineering approaches.

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.

Screening of GLA (γ-Linolenic Acid) from Fungi by Gas Chromatography and Mass Spectroscopy

In order to select acid (GLA)-producing fungi, a total of forty-four strains of 4 genera such as Phytophthora, Pythium, Mucor and Rhizopus were obtained from Koran Agricultural Culture Collection (KACC) and then analysed by using GC-FID and GC-MS. GLA was detected on 39 fungal strains, and the highest rate of GLA was found as 24.8% of total fatty acids on Mucor hiemalis f. sp. hiemalis KACC 40264. Total GLA content of Zygomycota was comparatively high - Mucor (14.2%) and Rhizopus (14.3%), whereas that of Oomycetes was low - Phytophthora (3.3%) and Pythium (3.0%). Moreover, total fatty acids of the Zygomycota fungi such as Mucor (15.4 mg/100 ml) and Rhizopus (7.1 mg/100 ml) were higher compared with the Oomycetes such as Phytophthora (2.6 mg/100 ml) and Pythium (4.5 mg/100 ml). Thus, two genera such as Mucor and Rhizopus have higher potential as an useful microbial resource. The total fatty acid content varies even within the strains of the same genus e.g. Mucor. M. blumbeus KACC 40935 showed the highest values on productivity (18.2%) of GLA and total fatty acid contents (50.8 mg/100 ml liquid medium).

Production of Hydroxymethylfurfrual by Sesamum indicum L. Root Cultures

Recently, hydroxymethylfurfrual (HMF) has been highlighted as a key intermediate for the production of liquid biofuels and other valuable compounds. We used sesame roots as a biocatalyst to synthesize HMF using flask cultures. The synthesis of HMF was identified by GC-mass analysis. The highest root growth was observed in cultures with 1.0 mg/l NAA at , while root growth was not found in those without NAA treatment. When silver nitrate () was added, the root growth was greatest in those treated with 0.5 mg/l and cultured at . In the case of HMF synthesis, its highest yield was obtained in those treated with 0.5 mg/l NAA at , but low HMF was detected in those treated without naphthaleneacetic acid (NAA). The addition of to the culture medium showed a 8-10% reduction in HMF yield compared to that of the control, indicating its inhibitory effect on the synthesis of HMF. On the whole, an optimal culture temperature for HMF synthesis seemed to be between .

Recent Studies of Edible Plant Vaccine for Prophylactic Medicine against Virus-mediated Diseases

Transgenic plants have been studied as delivery system for edible vaccine against various diseases. Edible plant vaccines have several potential advantages as follows: an inexpensive source of antigen, easy administration, reduced need for medical personnel, economical to mass produce and easy transport, heat-stable vaccine without refrigerator, generation of systemic and mucosal immunity and safe antigen without fetal animal-virus contaminants. The amount of recombinant antigens in transgenic plants ranged from 0.002 to 0.8％ in total soluble protein, depending on promoters for the expression of interested genes and plants to be used for transformation. Throughout the last decade, edible plant vaccine made notable progresses that protect from challenges against virus or bacteria. However edible plant vaccines have still problems that could be solved. First, the strong promoter or inducible promoter or strategy of protein targeting could be solved to improve the low expression of antigens in transgenic plants. Second, the transformation technique of target plant should be developed to be able to eat uncooked. Third, marker-free vector could be constructed to be more safety. In this review we describe advances of edible plant vaccines, focusing on the yields depending on plants/promoters employed and the results of animal/clinical trials, and consider further research for the development of a new plant-derived vaccine.

Essential arginyl and histidyl residues at the active site of NADP-malate dehydrogenase from Pisum sativum L. Giant leaves

Summary Chloroplast NADP-malate dehydrogenase from Pisum sativum L. Giant leaves was purified to homogeneity and chemically modified with various reagents. The enzyme activity was considerably decreased by 2,3-butanedione, diethylpyrocarbonate, and iodoacetamide. The inactivation of the enzyme by 2,3-butane-nedione or diethylpyrocarbonate was dependent on time and reagent concentration. The reaction orders with respect to these reagents were 1.08 and 1.25, respectively. The inactivation by 1 mmol/L 2,3-butanedione was selectively and considerably protected by addition of 10 mmol/L oxaloacetate, to the extent of a 4-fold decrease of inactivation. The inactivation by diethylpyrocarbonate was selectively prevented by 10 mmol/L NADPH. The inactivated enzyme by diethylpyrocarbonate was almost completely restored by incubation with 20 mmol/L hydroxylamine. The reduced enzyme, rather than the oxidized enzyme, was more susceptible to the inactivation using diethylpyrocarbonate or 2,3-butanedione. These results indicate that essential arginyl and histidyl residues are located at the active site of the enzyme.