Sun-Hyung Lim

Auto-excision of selectable marker genes from transgenic tobacco via a stress inducible FLP/FRT site-specific recombination system

Antibiotic resistance marker genes are powerful selection tools for use in plant transformation processes. However, once transformation is accomplished, the presence of these resistance genes is no longer necessary and can even be undesirable. We herein describe the successful excision of antibiotic resistance genes from transgenic plants via the use of an oxidative stress-inducible FLP gene. FLP encodes a recombinase that can eliminate FLP and hpt selection genes flanked by two FRT sites. During a transformation procedure in tobacco, transformants were obtained by selection on hygromycin media. Regenerants of the initial transformants were screened for selective marker excision in hydrogen peroxide supplemented media and both the FLP and hpt genes were found to have been eliminated. About 13–41% of regenerated shoots on hydrogen peroxide media were marker-free. This auto-excision system, mediated by the oxidative stress-inducible FLP/FRT system to eliminate a selectable marker gene can be very readily adopted and used to efficiently generate marker-free transgenic plants.

Combined Mass Spectrometry-Based Metabolite Profiling of Different Pigmented Rice (Oryza sativa L.) Seeds and Correlation with Antioxidant Activities

Nine varieties of pigmented rice (Oryza sativa L.) seeds that were black, red, or white were used to perform metabolite profiling by using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and gas chromatography (GC) TOF-MS, to measure antioxidant activities. Clear grouping patterns determined by the color of the rice seeds were identified in principle component analysis (PCA) derived from UPLC-Q-TOF-MS. Cyanidin-3-glucoside, peonidin-3-glucoside, proanthocyanidin dimer, proanthocyanidin trimer, apigenin-6-C-glugosyl-8-C-arabiboside, tricin-O-rhamnoside-O-hexoside, and lipids were identified as significantly different secondary metabolites. In PCA score plots derived from GC-TOF-MS, Jakwangdo (JKD) and Ilpoom (IP) species were discriminated from the other rice seeds by PC1 and PC2. Valine, phenylalanine, adenosine, pyruvate, nicotinic acid, succinic acid, maleic acid, malonic acid, gluconic acid, xylose, fructose, glucose, maltose, and myo-inositol were significantly different primary metabolites in JKD species, while GABA, asparagine, xylitol, and sucrose were significantly distributed in IP species. Analysis of antioxidant activities revealed that black and red rice seeds had higher activity than white rice seeds. Cyanidin-3-glucoside, peonidin-3-glucoside, proanthocyanidin dimers, proanthocyanidin trimers, and catechin were highly correlated with antioxidant activities, and were more plentiful in black and red rice seeds. These results are expected to provide valuable information that could help improve and develop rice-breeding techniques.

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.

Metabolite profiling approach reveals the interface of primary and secondary metabolism in colored cauliflowers (Brassica oleracea L. ssp. botrytis).

In the present study, carotenoids, anthocyanins, and phenolic acids of cauliflowers ( Brassica oleracea L. ssp. botrytis) with various colored florets (white, yellow, green, and purple) were characterized to determine their phytochemical diversity. Additionally, 48 metabolites comprising amino acids, organic acids, sugars, and sugar alcohols were identified using gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). Carotenoid content was considerably higher in green cauliflower; anthocyanins were detected only in purple cauliflower. Phenolic acids were higher in both green and purple cauliflower. Results of partial least-squares discriminant, Pearson correlation, and hierarchical clustering analyses showed that green cauliflower is distinct on the basis of the high levels of amino acids and clusters derived from common or closely related biochemical pathways. These results suggest that GC-TOFMS-based metabolite profiling, combined with chemometrics, is a useful tool for determining phenotypic variation and identifying metabolic networks connecting primary and secondary metabolism.

Unraveling the Light-Specific Metabolic and Regulatory Signatures of Rice through Combined in Silico Modeling and Multiomics Analysis

Combined in silico modeling and multiomics data analysis elucidate the transcriptional control of rice cellular metabolism upon light signaling. Light quality is an important signaling component upon which plants orchestrate various morphological processes, including seed germination and seedling photomorphogenesis. However, it is still unclear how plants, especially food crops, sense various light qualities and modulate their cellular growth and other developmental processes. Therefore, in this work, we initially profiled the transcripts of a model crop, rice (Oryza sativa), under four different light treatments (blue, green, red, and white) as well as in the dark. Concurrently, we reconstructed a fully compartmentalized genome-scale metabolic model of rice cells, iOS2164, containing 2,164 unique genes, 2,283 reactions, and 1,999 metabolites. We then combined the model with transcriptome profiles to elucidate the light-specific transcriptional signatures of rice metabolism. Clearly, light signals mediated rice gene expressions, differentially regulating numerous metabolic pathways: photosynthesis and secondary metabolism were up-regulated in blue light, whereas reserve carbohydrates degradation was pronounced in the dark. The topological analysis of gene expression data with the rice genome-scale metabolic model further uncovered that phytohormones, such as abscisate, ethylene, gibberellin, and jasmonate, are the key biomarkers of light-mediated regulation, and subsequent analysis of the associated genes’ promoter regions identified several light-specific transcription factors. Finally, the transcriptional control of rice metabolism by red and blue light signals was assessed by integrating the transcriptome and metabolome data with constraint-based modeling. The biological insights gained from this integrative systems biology approach offer several potential applications, such as improving the agronomic traits of food crops and designing light-specific synthetic gene circuits in microbial and mammalian systems.

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.

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.

Marker development for the identification of rice seed color

Pigmented traits in rice seeds are regarded as important breeding goals for crop improvement. Marker-assisted selection is very helpful when screening for target seed color traits in the early stages of plant development. Among the genes involved in the biosynthesis of anthocyanins and proanthocyanins (PAs) that are candidates for marker development, we examined the expression of five genes encoding CHS, CHI, F3H, DFR, and ANS in the seeds of non-pigmented white and pigmented black and red rice cultivars. The transcript levels of all these genes except for CHI are higher in pigmented rice than in non-pigmented rice. Sequence variations in these biosynthetic genes revealed that the DFR gene harbors a single nucleotide substitution that generates a premature stop codon in white rice. Additional sequence variations in two regulatory genes, OSB1 and Rc, were also compared among the same cultivars. The sequence of the OSB1 gene in black rice was found to differ from that in red and white rice. The sequence of the Rc gene in red rice also differed from that in white and black rice. Based on these variations, we developed two CAPS markers for DFR and OSB1 genes and an Indel marker for the Rc gene. The combined use of these three markers could discriminate rice seeds harboring white, black and red color. We validated the usefulness of these markers in 34 rice cultivars. Hence, the combined application of our three new markers may have utility to screen the seed color prior to seed setting in rice breeding programs.

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.

Identification and quantification of carotenoids in paprika fruits and cabbage, kale, and lettuce leaves

Twelve carotenoids were identified in Korean leafy vegetables and paprikas. by high-performance liquid chromatography, Carotenoid contents varied greatly, with red paprika having a higher antheraxanthin and capsanthin contents than other paprikas. Orange paprika had higher levels of zeaxanthin, β-cryptoxanthin, lutein, and α-carotene compared to those of other paprikas. The results of Pearson’s correlation analysis using quantitative data of carotenoids revealed that significant positive relationships were apparent between capsanthin and antheraxanthin (r=0.9870, p <0.0001), zeaxanthin and α-cryptoxanthin (r=0.9951, p <0.0001), as well as lutein and α-carotene (r=0.9612, p <0.0001). Because the correlations between carotenoids levels have provided valuable information regarding metabolic associations, this technique will contribute to identifying metabolic links for carotenoid biosynthesis.