Jae Kwang Kim

Carotenoid content and expression of phytoene synthase and phytoene desaturase genes in bitter melon (Momordica charantia)

Momordica charantia, a tropical plant, produces a fruit that has a β-carotene concentration five times higher than that of carrot. To elucidate the molecular basis of β-carotene accumulation in M. charantia, the gene expression levels of phytoene synthase (McPSY) and phytoene desaturase (McPDS) were determined. These levels were particularly high in the flowers of M. charantia. During fruit maturation, the expression levels of McPSY and McPDS decreased during the mid-stages but increased in the fully mature fruit. In addition, carotenoids accumulated as the peel changed from green to orange. Thus, McPSY and McPDS expression correlated with carotenoid accumulation during fruit maturation. Principal component analysis (PCA) also was used to evaluate the differences among the profiles of seven carotenoids identified in the fruit at several maturation stages. Riper fruits had higher carotenoid concentrations than less ripe fruits.

Genetic Modification of the Soybean to Enhance the β-Carotene Content through Seed-Specific Expression

The carotenoid biosynthetic pathway was genetically manipulated using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). The PAC gene was linked to either the β-conglycinin (β) or CaMV-35S (35S) promoter to generate β-PAC and 35S-PAC constructs, respectively. A total of 37 transgenic lines (19 for β-PAC and 18 for 35S-PAC) were obtained through Agrobacterium-mediated transformation using the modified half-seed method. The multi-copy insertion of the transgene was determined by genomic Southern blot analysis. Four lines for β-PAC were selected by visual inspection to confirm an orange endosperm, which was not found in the seeds of the 35S-PAC lines. The strong expression of PAC gene was detected in the seeds of the β-PAC lines and in the leaves of the 35S-PAC lines by RT-PCR and qRT-PCR analyses, suggesting that these two different promoters function distinctively. HPLC analysis of the seeds and leaves of the T2 generation plants revealed that the best line among the β-PAC transgenic seeds accumulated 146 µg/g of total carotenoids (approximately 62-fold higher than non-transgenic seeds), of which 112 µg/g (77%) was β-carotene. In contrast, the level and composition of the leaf carotenoids showed little difference between transgenic and non-transgenic soybean plants. We have therefore demonstrated the production of a high β-carotene soybean through the seed-specific overexpression of two carotenoid biosynthetic genes, Capsicum phytoene synthase and Pantoea carotene desaturase. This nutritional enhancement of soybean seeds through the elevation of the provitamin A content to produce biofortified food may have practical health benefits in the future in both humans and livestock.

Unintended polar metabolite profiling of carotenoid-biofortified transgenic rice reveals substantial equivalence to its non-transgenic counterpart

Substantial equivalence is a critical concept for biosafety assessment of genetically modified (GM) crops. To investigate substantial equivalence among carotenoid-biofortified GM rice and five conventional rice cultivars having common white (three) and red (two) grain colors, profiles of 52 polar metabolites were analyzed using gas chromatography time-of-flight mass spectrometry. The results were compared to evaluate the differences among GM and non-GM rice cultivars using principal components analysis. The GM rice is more comparable to its non-transgenic counterpart rice variety according to the closer co-separation than for other cultivars tested. This suggests that profiling of unintended polar metabolites could be a useful tool to reveal substantial equivalence of GM rice.

Metabolomics analysis and biosynthesis of rosmarinic acid in Agastache rugosa Kuntze treated with methyl jasmonate.

This study investigated the effect of methyl jasmonate (MeJA) on metabolic profiles and rosmarinic acid (RA) biosynthesis in cell cultures of Agastache rugosa Kuntze. Transcript levels of phenylpropanoid biosynthetic genes, i.e., ArPAL, Ar4CL, and ArC4H, maximally increased 4.5-fold, 3.4-fold, and 3.5-fold, respectively, compared with the untreated controls, and the culture contained relatively high amounts of RA after exposure of cells to 50 µM MeJA. RA levels were 2.1-, 4.7-, and 3.9-fold higher after exposure to 10, 50, and 100 µM MeJA, respectively, than those in untreated controls. In addition, the transcript levels of genes attained maximum levels at different time points after the initial exposure. The transcript levels of ArC4H and Ar4CL were transiently induced by MeJA, and reached a maximum of up to 8-fold at 3 hr and 6 hr, respectively. The relationships between primary metabolites and phenolic acids in cell cultures of A. rugosa treated with MeJA were analyzed by gas chromatography coupled with time-of-flight mass spectrometry. In total, 45 metabolites, including 41 primary metabolites and 4 phenolic acids, were identified from A. rugosa. Metabolite profiles were subjected to partial least square-discriminate analysis to evaluate the effects of MeJA. The results indicate that both phenolic acids and precursors for the phenylpropanoid biosynthetic pathway, such as aromatic amino acids and shikimate, were induced as a response to MeJA treatment. Therefore, MeJA appears to have an important impact on RA accumulation, and the increased RA accumulation in the treated cells might be due to activation of the phenylpropanoid genes ArPAL, ArC4H, and Ar4CL.

Compositional comparative analysis between insect-resistant rice (Oryza sativa L.) with a synthetic cry1Ac gene and its non-transgenic counterpart

Composition analysis of genetically modified crops is an important consideration in the assessment of food safety. Agb0101 (Oryza sativa L. cv. Nakdongbyeo) developed in Korea is a form of insect-resistant rice that contains a synthetic truncated cry1Ac gene isolated from Bacillus thuringiensis (Bt), and demonstrates high resistance to rice leaf folder under field conditions. Nutrients, anti-nutritive components, and secondary metabolites of Agb0101 were analyzed and compared with those of its non-transgenic counterpart. The amounts of proximates, amino acids, fatty acids, minerals, vitamins, trypsin inhibitors, phytic acid, and ferulic acid in brown rice from Agb0101 were comparable to those of its non-transgenic counterpart. Statistical comparisons to test for equivalence showed that all the analyzed components in the insect-resistant rice plants were substantially equivalent to those of its non-transgenic counterpart. Furthermore, most of the measured values from Agb0101 were within the range of values reported for other commercial rice varieties.

Vascular-specific activity of the Arabidopsis carotenoid cleavage dioxygenase 7 gene promoter

Carotenoid cleavage dioxygenases (CCDs) are involved in the production of diverse apocarotenoids including phytohormones, the visual molecules and the aromatic volatile compounds derived from carotenoids. Here, we examined the spatial expression of four of the CCD genes (AtCcd1, 4, 7 and 8) among the nine members of this family in Arabidopsis by RT-PCR. We found that the AtCcd7 gene showed strong expression in seeds. However, the promoter activity of the 1,867-bp 5′-upstream region of this gene exhibited a vascular specificity at all developmental stages throughout the transgenic Arabidopsis plants tested. The strength of the AtCcd7 promoter was also found to be lower than that of the 35S promoter by about 60%. The whole body expression of the β-glucuronidase (GUS) reporter gene driven by the AtCcd7 promoter in Arabidopsis plants was confirmed in different organs by RT-PCR and GUS enzymatic assays. Histochemical GUS staining further revealed that the AtCcd7 promoter has utility in limiting the expression of target genes to the vascular tissues in all plant organs such as the leaf, stem, root, flower and seed.

Metabolomics for the Quality Assessment of Lycium chinense Fruits

Lycium chinense has been used as a traditional medicine for centuries in Asia because of its positive effects on health. However, its functional components have not been elucidated. This study determines the levels of health-promoting lipophilic compounds, including carotenoids, tocopherols, and phytosterol, and those of 42 hydrophilic metabolites, including sugars, organic acids, alcohols, amines, and amino acids, in L. chinense fruit from 11 cultivars. The metabolite profiles were subjected to a principal component analysis (PCA), Pearson correlation analysis, and hierarchical clustering analysis (HCA). PCA showed the Cheongdang (LM-3) cultivar to be distinct from the others. The correlation results for a total of 55 compounds revealed strong correlations between the metabolites that participated on closely related pathways. The Cheongdang cultivar appears to be most suited for functional food production because of its high carotenoid, tocopherol, and phytosterol levels. These results indicate the usefulness of metabolite profiling as a tool for assessing the quality of food.

Variation and correlation analysis of phenolic compounds in mungbean (Vigna radiata L.) varieties

Phenolic compounds from a wide collection of mungbean [Vigna radiata (L.) Wilczek] germplasm (56 varieties) were characterised to determine the diversity among these phytochemicals and to analyse the relationships among their contents. The profiles of 25 phenolic compounds identified from the grains were subjected to data-mining processes, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), Pearsons correlation analysis, and hierarchical clustering analysis (HCA). The IT212105 and IT104818 varieties separated from the others in the first two principal components of PCA. PLS-DA showed significant separation between extracts of mungbean originating from three countries: China, Japan, and Korea. HCA of these phytochemicals resulted in clusters derived from common or closely related biochemical pathways. Significant positive relationships were observed between coumaric acid and resveratrol (r=0.7195, p<0.0001). Catechin content was positively correlated with rutin (r=0.6291, p<0.0001). The IT104818 variety appears to be a good candidate for future breeding programs, as it contains high levels of phenolic compounds. These results demonstrate the use of metabolic profiling combined with chemometrics as a tool for assessing the quality of food.

Use of an anthocyanin production phenotype as a visible selection marker system in transgenic tobacco plant

To develop a potentially alternative method for the selection of transgenic plants instead of antibiotic and herbicide resistance, anthocyanin pigmentation phenotype was examined to provide a visible selection marker system. Two regulatory genes of the anthocyanin biosynthetic pathway, the R2R3 MYB mPAP1 gene from Arabidopsis and the basic helix loop helix B-Peru gene from maize, were amplified by RT-PCR and then individually cloned into a plant expression vector. The requirement of these two genes for anthocyanin pigmentation was pre-confirmed via an in vivo assay using tobacco agro-infiltration. The mPAP1 and B-Peru vectors were further stably co-transformed into tobacco plants using Agrobacterium tumefaciens strain LBA4404. Tobacco plants harboring both genes could be readily selected through the manifestation of a red color due to anthocyanin accumulation in the whole plant body. The T1 segregants showed red or green phenotypes depending on the genotype. The need for both the mPAP1 and B-Peru genes for a red color phenotype due to anthocyanin pigmentation was further confirmed by genotyping of the T1 generation by genomic PCR analysis and an in vivo assay using agro-infiltration. From these results, we conclude that co-transformation with two individual vectors harboring a critical anthocyanin transcriptional factor has potential utility as an alternative visible selectable marker system for transgenic progeny selection in plants.

Determination of phenolic acids in Korean rice (Oryza sativa L.) cultivars using gas chromatography-time-of-flight mass spectrometry

Total soluble phenolic acids (free and esterified forms) from 15 Korean rice cultivars (Oryza sativa L.) were characterized to determine the total soluble phenolic acid contents and the diversity among the phenolic acids. The individual phenolic acids were identified by GC coupled to time-of-flight MS (TOFMS) including tertbutyldimethylsilyl (TBDMS) derivatization. This method requires only 13 min for the GC-TOFMS run. Total soluble phenolic acid contents were higher in pigmented rice (168–368 μg/g) than nonpigmented rice (146–172 μg/g). Freeform phenolic acids accounted for less than 10% of the total soluble phenolic acids. The dominant compounds were ferulic and sinapic acid, which were detected as cisand trans-isomers. The content of salicylic acid in rice grain varied, ranging from 7.95 to 29.61 μg/g. The quantitative results of this study indicate that the black rice ‘Josengheugchalbyeo’ could facilitate development of phenolic-rich rice or food.