Si-Myung Lee

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.

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.

A Sepal-Expressed ADP-Glucose Pyrophosphorylase Gene (NtAGP) Is Required for Petal Expansion Growth in ‘Xanthi’ Tobacco

In this study, a tobacco (Nicotiana tabacum ‘Xanthi’) ADP-glucose pyrophosphorylase cDNA (NtAGP) was isolated from a flower bud cDNA library and the role of NtAGP in the growth of the floral organ was characterized. The expression of NtAGP was high in the sepal, moderate in the carpel and stamen, and low in the petal tissues. NtAGP-antisense plants produced flowers with abnormal petal limbs due to the early termination of the expansion growth of the petal limbs between the corolla lobes. Microscopic observation of the limb region revealed that cell expansion was limited in NtAGP-antisense plants but that cell numbers remained unchanged. mRNA levels of NtAGP, ADP-glucose pyrophosphorylase activity, and starch content in the sepal tissues of NtAGP-antisense plants were reduced, resulting in significantly lower levels of sugars (sucrose, glucose, and fructose) in the petal limbs. The feeding of these sugars to flower buds of the NtAGP-antisense plants restored the expansion growth in the limb area between the corolla lobes. Expansion growth of the petal limb between the corolla lobes was severely arrested in ‘Xanthi’ flowers from which sepals were removed, indicating that sepal carbohydrates are essential for petal limb expansion growth. These results demonstrate that NtAGP plays a crucial role in the morphogenesis of petal limbs in ‘Xanthi’ through the synthesis of starch, which is the main carbohydrate source for expansion growth of petal limbs, in sepal tissues.

Characterization of the potato upreg1gene, encoding a mutated ADP-glucose pyrophosphorylase large subunit, in transformed rice

The potato upreg1, which encodes a mutated ADP-glucose pyrophosphorylase (AGPase) large subunit, was introduced into rice to evaluate its potential to enhance sink-driven yield productivity in this crop. We also wished to elucidate the activities of the up-regulated allosteric variants of potato AGPase large subunit gene in rice. A T-DNA vector containing the upreg1 gene under the control of the rice glutelin promoter was constructed with a MAR sequence and transformed into rice using Agrobacterium-mediated transformation. Transgenic plants were selected on medium supplemented with phosphinothricin and confirmed by the application of herbicide. A total of 38 transgenic plants were subsequently obtained in which the integration upreg1 into the rice genome was confirmed by Southern blotting. The exogenous AGPase in transgenic rice plants showed a high affinity for 3-phosphoglycerate activator and a low affinity for the orthophosphate inhibitor, as observed in lettuce. The transgenic rice also showed increases in the number of grains per particle, the number of panicles per plant, and also in the fresh weight of the above-ground mass of plant which was about 15% higher than non-transgenic ‘Nak-dong’. The number of seeds per tiller was also found to be about 10% higher in the transgenic plants. However, the net photosynthesis rate showed very little difference in the transgenic rice, and we could not therefore confirm any linkage with the deregulation of allosteric effects. Based on these results, upreg1 mutant genes can be used for the genetic improvement of plant AGPases other than potato and to effectively increase crop yield productivity.

Proteomic identification of a novel toxin protein (Txp40) from Xenorhabdus nematophila and its insecticidal activity against larvae of Plutella xylostella.

For the identification of a novel insecticidal protein, a two-dimensional liquid chromatography (PF-2D) system was used in a quantitative proteomic analysis of Xenorhabdus nematophila CBNU strain isolated from entomophagous nematode Steinernema carpocapsae . Protein patterns obtained from minimum and maximum insecticidal activities during cultivation were contrasted, and a novel toxin protein (Txp40) was identified by MALDI-TOF/MS. The DNA sequence of the cloned toxin gene (1089 bp) has an open reading frame encoding 363 amino acids with a predicted molecular mass of 41162 Da. The txp40 identified in this study is most closely related to the known txp40 cloned from X. nematophila EB (ADQ92844) with 94.4% identical sequence residues. Following the expression of the newly identified toxin gene in Escherichia coli , the insecticidal activity of the recombinant toxin protein was determined against Plutella xylostella larvae; a 56.7% mortality rate was observed within 24 h.

D-amino Acid Oxidase (DAO) Gene as a Novel Selection Marker for Plant Transformation

Though higher plants car not metabolize D-amino acid, many prokaryotes and eukaryotes have the D-amino acid metabolism. Therefore, we transformed tobacco plants with D-amino acid oxidase (DAO), which can metabolize D-amino acid, and confirmed that transgenic tobacco plants might metabolize D-amino acid. Transgenic tobacco plants were survived a high concentration of D-serine, however non-transgenic plants were not grown on D-serine medium. From Southern and Northern blot analysis, transgenic tobacco plants selected on D-serine medium were confirmed by insert and expression of transgene. tobacco seeds derived tobacco plants selfing were grown on D-serine medium and showed normal phenotype compared to wild tobacco plants. Transgenic tobacco plants displayed the metabolic capability of D-serine. Therefore, we suggested that DAO is useful selectable marker gene for plant transformation.

Molecular Characterization and Event-Specific Marker Development of Insect Resistant Chinese Cabbage for Environmental Risk Assessment

Commercialization of genetically modified (GM) plants will be required the assessment of risks associated with the release of GM plants that should include a detailed risk assessment of their impacts in human health and the environment. Prior to GM plant release, applicants should provide the information on GM crops for approval. We carried out this study to provide the molecular data for risk assessment of the GM Chinese cabbage plants with insect-resistance gene, modified CryIAc, which we obtained by Agrobacterium-transformation. From the molecular analysis with GM Chinese cabbage, we confirmed the transgene copy number and stability, the expression of the transgene, and integration region sequences between the transgene and the Chinese cabbage genome. Based on the unique integration DNA sequences, we designed specific primer set to detect GM Chinese cabbage and set up the GM cabbage detection method by qualitative PCR analysis. Qualitative analysis with GM Chinese cabbage progenies analysis was revealed the same as the result of herbicide treatment. Our results provided the molecular data for risk assessment analysis of GM Chinese cabbage and demonstrated that the primer set proposed could be useful to detect GM Chinese cabbage.

Influence of Cooking on Nutrient Composition in Provitamin A- Biofortified Rice

This study was conducted to investigate the effects of different cooking methods on the nutrient composition of genetically modified (GM) provitamin A(PA)-biofortified rice. PA-biofortified rice was subjected to different cooking methods, namely boiling and a soaking, steaming and roasting process. The proximate components (starch, protein, lipid and ash) of raw PA-biofortified rice were similar to those found in the parental non-GM rice, and were not significantly affected by a conventional boiling treatment. When compared with raw rice, boiled PA-biofortified rice showed a similar nutrient composition, despite a slight reduction in a majority of its amino acid contents. However, the PA-biofortified rice that underwent the soaking/steaming/roasting process exhibited a significant reduction in a majority of its amino acids and mineral contents. This procedure also led to a significant reduction in carotenoid contents. The overall results of this study demonstrate that using a conventional boiling method for PA-biofortified rice retains nutrients better than a soaking/ steaming/roasting method.

Analysis of junction between T-DNA and plant genome in insect resistance GM Chinese cabbage

The Agrobacterium-mediated transformation has been successfully used method to introduce foreign genes into some monocotyledonous as well as a large number of dicotyledonous plants genome, We developed transgenic Chinese cabbage plants with insect-resistance gene, modified CryIAc, by Agrobacterium-transformation and confirmed transgene copy number by Southern blot analysis. We confirmed that twenty-nine out of 46 transgenic Chinese cabbage plants have single copy of CryIAc. To obtain the sequences information on the transferred DNA (T-DNA) integration into plant genome, we analyzed left border (LB) flanking sequences by genome walking (GW) PCR method. Out of 46 transgenic Chinese cabbage plants examined, 37 carried the vector backbone sequences. This result indicates that the transfer of the vector backbone from the binary vectors resulted mainly from inefficient termination of LB site. Analysis of T-DNA LB flanking region of 9 transgenic Chinese cabbage plants without vector backbone revealed that all LB ends were not conserved and nucleotides up to 36bp from the LB cleavage site were deleted.

Comparative evaluation of nutritional compositions between transgenic rice harboring the CaMsrB2 gene and the conventional counterpart

As a part of a safety assessment of new transgenic crops, compositional equivalence studies between transgenic crops with non-transgenic comparators are almost universally required. This study was conducted to compare nutritional profiles of proximate composition, and fatty acid, amino acid, mineral, and vitamin contents, and anti-nutrients, between transgenic drought-tolerant Agb0103 rice harboring the pepper methionine sulfoxide reductase B2 gene CaMsrB2 and the parental rice cultivar, ‘Ilmi’ as a non-transgenic control. Both transgenic and non-transgenic rice were grown and harvested in 2 different locations. Proximate compositions of moisture, starch, protein, lipid, and ash content of Agb0103 rice were similar to parental non-transgenic rice. There were no differences between transgenic and non-transgenic rice with respect to the whole nutritional composition, except for minor locality differences for a few nutritional components. Agb0103 rice with improved resistance to drought is nutritionally equivalent to the parental rice cultivar.