Jong-Sug Park

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.

Exploiting leaf starch synthesis as a transient sink to elevate photosynthesis, plant productivity and yields.

Improvements in plant productivity (biomass) and yield have centered on increasing the efficiency of leaf CO(2) fixation and utilization of products by non-photosynthetic sink organs. We had previously demonstrated a correlation between photosynthetic capacity, plant growth, and the extent of leaf starch synthesis utilizing starch-deficient mutants. This finding suggested that leaf starch is used as a transient photosynthetic sink to recycle inorganic phosphate and, in turn, maximize photosynthesis. To test this hypothesis, Arabidopsis thaliana and rice (Oryza sativa L.) lines were generated with enhanced capacity to make leaf starch with minimal impact on carbon partitioning to sucrose. The Arabidopsis engineered plants exhibited enhanced photosynthetic capacity; this translated into increased growth and biomass. These enhanced phenotypes were displayed by similarly engineered rice lines. Manipulation of leaf starch is a viable alternative strategy to increase photosynthesis and, in turn, the growth and yields of crop and bioenergy plants.

Isolation and functional characterization of polyunsaturated fatty acid elongase (AsELOVL5) gene from black seabream (Acanthopagrus schlegelii)

To identify the genes encoding fatty acid elongases for the biosynthesis of polyunsaturated fatty acids (PUFAs), we isolated a cDNA via degenerate PCR and RACE-PCR from Acanthopagrus schlegelii with a high similarity to the ELOVL5-like elongases of mammals and fishes. This gene is termed AsELOVL5 and encodes a 294 amino acid protein. When AsELOVL5 was expressed in Saccharomyces cerevisiae, it conferred an ability to elongate γ-linolenic acid (18:3 n−6) to di-homo-γ-linolenic acid (20:3 n−6). In addition, the transformed cells converted arachidonic acid (20:4 n−6) and eicosapentaenpic acid (20:5 n−3) to docosatetraenoic acid (22:4 n−6) and docosapentaenoic acid (22:5 n−3), respectively. These results indicate that the AsELOVL5 gene encodes a long-chain fatty acid elongase capable of elongating C18Δ6/C20Δ5 but not C22 PUFA substrates.

A pepper MSRB2 gene confers drought tolerance in rice through the protection of chloroplast-targeted genes.

Background The perturbation of the steady state of reactive oxygen species (ROS) due to biotic and abiotic stresses in a plant could lead to protein denaturation through the modification of amino acid residues, including the oxidation of methionine residues. Methionine sulfoxide reductases (MSRs) catalyze the reduction of methionine sulfoxide back to the methionine residue. To assess the role of this enzyme, we generated transgenic rice using a pepper CaMSRB2 gene under the control of the rice Rab21 (responsive to ABA protein 21) promoter with/without a selection marker, the bar gene. Results A drought resistance test on transgenic plants showed that CaMSRB2 confers drought tolerance to rice, as evidenced by less oxidative stress symptoms and a strengthened PSII quantum yield under stress conditions, and increased survival rate and chlorophyll index after the re-watering. The results from immunoblotting using a methionine sulfoxide antibody and nano-LC-MS/MS spectrometry suggest that porphobilinogen deaminase (PBGD), which is involved in chlorophyll synthesis, is a putative target of CaMSRB2. The oxidized methionine content of PBGD expressed in E. coli increased in the presence of H2O2, and the Met-95 and Met-227 residues of PBGD were reduced by CaMSRB2 in the presence of dithiothreitol (DTT). An expression profiling analysis of the overexpression lines also suggested that photosystems are less severely affected by drought stress. Conclusions Our results indicate that CaMSRB2 might play an important functional role in chloroplasts for conferring drought stress tolerance in rice.

Molecular Analysis of Microbial Community in Soils Cultivating Bt Chinese Cabbage

*(National Academy of Agricultural Science, Suwon, 441-707, Korea) The aim of this study was to investigate the possible impact of Bt Chinese cabbage on the soil microbial community. Microbial communities were isolated from the rhizosphere of one Bt Chinese cabbage variety and four varieties of conventional ones and were subjected to be analyzed using both culture-dependent and molecular methods. The total counts of bacteria, fungi, and actinomycetes in the rhizosphere of transgenic and conventional Chinese cabbages were observed to have an insignificant difference. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes revealed that the bacterial community structures were very similar to each other and this genetic stability of microbial communities was maintained throughout the culture periods. Analysis of dominant isolates in the rhizosphere of transgenic and conventional Chinese cabbages showed that the dominant isolates from the soil of transgenic Chinese cabbage belonged to the Bacilli and Alphaproteobacteria, while the dominant isolates from the soil of conventional cabbage belonged to the Holophagae and Planctomycetacia, respectively. These results indicate that the Bt transgenic cabbage has no significant impact on the soil microbial communities.

Isolation and functional characterization of a delta 6-desaturase gene from the pike eel (Muraenesox cinereus)

Stearidonic acid (STA; 18:4n-3) and γ-linolenic acid (GLA; 18:3n-6) are significant intermediates in the biosynthetic pathway for the very-long-chain polyunsaturated fatty acids of eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (ARA; 20:4n-6), respectively. To develop a sustainable system for the production of dietary polyunsaturated fatty acids, we focused on the action of the enzyme delta 6-desaturase (D6DES) on the essential acids, linoleic acid (LA; 18:2n-6) and α-linolenic acid (ALA; 18:3n-3). A 1,335-bp full-length cDNA encoding D6DES (McD6DES) was cloned from Muraenesox cinereus using degenerate PCR and RACE-PCR methods. To investigate the enzymatic activity of McD6DES in the production of n-6 and n-3 fatty acids, a recombinant plasmid expressing McD6DES (pYES-McD6DES) was transformed into and expressed in Saccharomyces cerevisiae. The exogenously expressed McD6DES produced GLA and STA at conversion rates of 14.2% and 45.9%, respectively, from the exogenous LA and ALA substrates. These results indicate that McD6DES is essentially a delta 6-desaturase involved in very-long-chain polyunsaturated fatty acid synthesis.

Proteomic identification of toxic volatile organic compound-responsive proteins in Arabidopsis thaliana

The proteins that are responsive to toxic volatile organic compounds (VOC) such as formaldehyde and toluene were analyzed with proteome analysis using two-dimensional difference image gel electrophoresis (DIGE) technology. Twenty-one days after germination (DAG) seedlings of Arabidopsis thaliana were exposed either to the gaseous formaldehyde or toluene in an airtight box installed in a plant growth chamber maintained at 24°C under the long day condition with relatively low light condition. Comparative expression analysis revealed 14 and 22 protein spots as proteins displaying at least 1.5-fold differences in expression upon formaldehyde and toluene treatment, respectively, compared to those of untreated control. Most of the isolated spots were successfully identified by peptide analysis using LC-MS-MS. The VOC-responsive proteins contain ATP synthase CF1, ribulose-1,5-bisphosphate carboxylase/oxygenase, photosystem II light harvesting complex, and enolase, which are components of photosynthesis and carbohydrate metabolism. Despite the relatively low light intensity was applied, many identified VOC-induced proteins were previously known to be up-regulated upon high light stimulus. In addition, proteins involved in the toxin catabolic process and stress hormone-related proteins were identified as toluene-induced proteins. Although the exact function of most of the VOC-responsive proteins identified in these experiments had not been characterized, the protein expression analysis using DIGE was clearly demonstrated that plants are capable of responding actively to VOCs at translational level, and identified proteins may provide valuable tools to account for the effects of abiotic stress caused by air pollutants such as VOCs in plant.

Recombinant DNA and Protein Vaccines for Foot-and-mouth Disease Induce Humoral and Cellular Immune Responses in Mice.

Background Foot-and-mouth disease virus (FMDV) is a small single-stranded RNA virus which belongs to the family Picornaviridae, genus Apthovirus. It is a principal cause of FMD which is highly contagious in livestock. In a wild type virus infection, infected animals usually elicit antibodies against structural and non-structural protein of FMDV. A structural protein, VP1, is involved in neutralization of virus particle, and has both B and T cell epitopes. A RNA-dependent RNA polymerase, 3D, is highly conserved among other serotypes and strongly immunogenic, therefore, we selected VP1 and 3D as vaccine targets. Methods VP1 and 3D genes were codon-optimized to enhance protein expression level and cloned into mammalian expression vector. To produce recombinant protein, VP1 and 3D genes were also cloned into pET vector. The VP1 and 3D DNA or proteins were co-immunized into 5 weeks old BALB/C mice. Results Antigen-specific serum antibody (Ab) responses were detected by Ab ELISA. Cellular immune response against VP1 and 3D was confirmed by ELISpot assay. Conclusion The results showed that all DNA- and protein-immunized groups induced cellular immune responses, suggesting that both DNA and recombinant protein vaccine administration efficiently induced Ag-specific humoral and cellular immune responses.

Carotenoids Biosynthesis and Their Metabolic Engineering in Plants

Carotenoids are synthesized from the plastidic glyceraldehyde-3-phosphate (GAP)/pyruvate pathway in isoprenoids biosynthetic system of plants. They play a crucial role in light harvesting, work as photoprotective agents in photosynthesis of nature, and are also responsible for the red, orange and yellow colors of fruits and flowers in plants. In addition to biological actions of carotenoids as antioxidants and natural pigments, they are essential components of human diet as a source of vitamin A. It has been also suggested that some kinds of carotenoids might provide protection against cancer and heart disease as human medicines. In this article, we review the commercial applications on the basis of biological functions of carotenoids, summarize the studies of genes involved in the carotenoid biosynthetic pathway, and introduce recent results achieved in metabolic engineering of carotenoids. This effort for understanding the carotenoids metabolism will make us to increase the total carotenoid contents of crop plants, direct the carotenoid biosynthetic machinery towards other useful carotenoids, and produce a new array of carotenoids by further metabolizing the new precursors that are created when one or two key enzymes in carotenoid biosynthetic pathway are exchanged through gene manipulation in the near future.