Dong-Hern Kim

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.

Kinetic and regulatory properties of plant ADP-glucose pyrophosphorylase genetically modified by heterologous expression of potato upreg mutants in vitro and in vivo

In this study, the uses of the mutated genes, upreg1 and upreg2, encoding upregulated ADP-glucose pyrophosphorylase (AGPase) large subunits with increased enzymatic activity, to improve crop yield productivity was evaluated inxa0vitro and inxa0planta. For inxa0vitro examination, wild type and upregs were co-expressed with three different AGPase small subunit genes from potato and perilla to produce nine AGPase isoforms. In kinetic experiments, 3-Phosphoglycerate increased the Vmax and decreased the KM for the recombinant AGPase. Regardless of the specific small subunit, Upreg-type AGPases had much larger increases in enzymatic activity with concomitant decreases in values as compared to the wild type enzyme. Transformation of lettuce with the upreg1 gene altered the regulatory properties of leaf AGPase. AGPases from transgenic lettuce showed greater 3-PGA activation and lower Pi inhibition than was observed for wild type AGPase. Fresh weights of the aerial parts of transgenic plants were larger than non-transgenic controls. Based on these results, upreg mutant genes could be used for the genetic improvement of plant AGPases other than potato and effectively increase crop yield productivity.

Petal-specific activity of the promoter of an anthocyanidin synthase gene of tobacco (Nicotiana tabacum L.)

Anthocyanidin synthase (ANS) is a key enzyme in the late stages of the biosynthesis of anthocyanins, an important class of plant pigments. Two ANS genes (NtANS1 and NtANS2) were cloned from flowers of the tobacco plant (Nicotiana tabacum). These genes exhibit similar genomic structures and are present as single-copy genes. Sequence analysis of the coding regions of the tobacco ANS genes has revealed high levels of amino acid identity with ANS proteins of other plants, with 70–87xa0% homology at the amino acid level. Analysis of the spatial regulation of expression of tobacco ANS genes has revealed that levels of their transcripts are particularly abundant in petal tissues, among all organs (leaf, stem, root, flower, and seed) and flower tissues (petal, pistil, stamen, and sepal) analyzed. Moreover, levels of gene expression increase with maturation, and then decrease slightly during the final stages of flower development. To investigate activity of the promoter of the NtANS1 gene, the cis-acting elements in the 955-nucleotide region upstream of its start codon are analyzed. Placing the expression of the gene that encodes β-glucuronidase (GUS) under the control of the NtANS1 promoter (NtANS1-P) in transgenic tobacco plants enables the authentic petal-specific expression of this gene to be verified by analysis of different flower tissues by histochemical GUS staining, semi-quantitative RT-PCR, and fluorometric GUS assays. In conclusion, given the ability of the NtANS1-P to restrict target gene expression to petals, it might have value in engineering desirable changes in petal color in the flowers of genetically modified plants.

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.

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.

Functional implications of gene expression analysis from rice tonoplast intrinsic proteins during seed germination and development

Rice seed maturation and germination involve drastic changes in water and nutrient transport, in which tonoplast aquaporins may play an important role. In the present study, gene expression profiles of 10 tonoplast intrinsic proteins (TIP) from rice were investigated by RT-PCR during seed development and germination. OsTIP3;1 and OsTIP3;2 were specifically expressed in mature seeds. Their transcript level rapidly decreased after onset of seed germination and gene expression was induced by ABA treatment. In contrast, expression of OsTIP2;1 and OsTIP4;3 was not seed specific as transcripts were found in vegetative tissues as well. Their respective transcript levels decreased at an early stage of seed development, whereas they increased at a later stage of seed germination and elongation of embryonic roots and shoots. When seed germination was inhibited by various stress conditions and ABA, expression of OsTIP2;1 and OsTIP4;3 was completely suppressed. In contrast, the expression level of OsTIP2;2 rapidly increased after seed imbibition and the transcript level was maintained under conditions inhibiting seed germination. These results implicate that tissue specific and developmental transcriptional regulation of OsTIPs in rice seeds depends on their specific function. In addition, OsTIPs can be discriminated by different potential phosphorylation and methylation sites in their protein structures. OsTIP3;1 and OsTIP3;2 possess unique phosphorylation signatures at their N-terminal domain, loop B and loop E, respectively. OsTIP2;1 and OsTIP4;3 have a potential methylation site at their Nterminal domain. This suggests that activity of specific tonoplast aquaporins may be regulated by post-translational modification as well as by transcriptional control.

Current status on carbon metabolic engineering in plants

Abstract Yield productivity of staple crops must be in-creased at least 50% by 2050, in order to feed the world population which is expected to reach 90 billions. Photo-synthetic carbon assimilation and carbohydrate metabolism leading to the production of starch would be the final frontier to quest for new sources of technology enabling such a dra-stic increase of crop productivity. In this review, attempts to genetically engineer plant photosynthetic carbon reduction cycle and metabolic pathways to increase starch production are introduced. Keywords ADP-glucose pyrophosphorylase, calvin cycle, flux control analysis, photosynthesis, yield productivity, transgenesis 서 론 금세기에 들어 작물의 생산성 향상은 새로운 화두로 대두되고 있다. 2007년과 2008년에는 유례가 없을 정도의 곡물가 상승이 일어나 중국 18% 세계 인구가 지속적으로 증가하여 2050년에는 90억명에 달할 것으로 예상되고 있으며 이들을 먹여 살리기 위해서는 현재 보다 50%정도의 곡물생산이 증가해야 한다. 또한 개발도상국의 경제 발전에 따른 육류 수요 증가와 이에 따른 사료 수요의 증가, 온실가스 방출억제와 지구온난화에 대응한 바이오에너지 원료 수요 증가 등 작물의 생산성 증가는 그 어느 때보다 절실한 실정이다. 식물의 광합성을 통해 생성된 탄수화물은 지구상의 생명체가 삶을 유지할 수 있는 기본적인 에너지원이다. 식물은 1차 생산자로서 공기 중의 이산화탄소를 유기태의 탄소화합물로 전환시키며, 이후 복잡한 대사 네트워크를 통해 다양한 생체 유기물질을 생산한다. 작물이 생산, 저장하는 전분, 지질 및 단백질 등은 농업생산성 측면에서 많은 연구자들의 관심을 끌어왔으며, 특히 유전자의 분리, 변형, 유전자전환 등 생명공학 기술이 발달함에 따라 작물의 탄소대사를 변형시켜 생산성을 증가시키기 위한 대사공학연구가 활발하게 진행되고 있다. 본 총설에서는 작물의 생산성과 광합성 및 탄소대사의 관계를 고찰하고 생명공학 기술을 이용한 작물 생산대사 형질 개선 연구현황에 대해 간략하게 소개하고자 한다. 작물의 광합성과 생산성 작물의 광합성은 대기중의 이산화탄소가 유기물의 형태로 전환되는 최초의 과정으로 종자 중의 전분 등 저장물질 생산도 잎의 광합성에 의해 생성된 탄수화물에서 유래된 것이다. 그러나 작물의 생산성과 광합성 대사활성간의 관계에 대해서는 논쟁의 여지가 있다. 일례로 밀 재배품종과 야생종의 광합성을 비교한 실험의 결과를 보면 재배품종의 광합성 활성이 야생종에 비해 낮았다는 Evans와 Dunstone (1970)의 보고 이외에도 작물의 생산성과 광합성 간에 상관관계가 없다는 연구 결과들은 상당히 많이 있다 (Evans 1993, 1998). 그러나 호주의 빵밀 계통에 국한하여 작물의 생산성과 광합성을 조사한 결과 Evans와 Dunstone의 초기 실험과는 반대로 정의 상관관계를 보여 유전적 다양성이 적은 품종간의 광합성 차이와

Characteristics and functions of shaker like potassium channels in rice

Abstract Potassium (K + ) is one of the most abundant cations in higher plant. It comprises about 10% of plant dry weight and it plays roles in numerous functions such as osmo- and turgor regulation, charge balance of plasma mem-brane and control of stomata and organ movement. Several potassium transporters and potassium channels regulate K + homeostasis in response to K + uptake systems. In this review, we describe the biological, biochemical and physiological characteristics of shaker like potassium channels in higher plant. Especially, we searched the rice genome databases and analysized expressed genes, genome structures and protein domain characteristics of shaker like potassium channels. Keywords plant potassium channel, shaker like, rice 서 론 식물 생장에 필수적인 무기 영양소 중 K + (potassium) 이온은 식물체 내에서 가장 풍부한 양이온으로서 조직이나 식물에 따라 다르나 mM 수준의 농도로 식물 세포에 존재한다. K + 이온은 호흡과 광합성에 필요한 효소들의 활성조절, 세포막 전위차 (plasmamembrane potential)의 조절, 그리고 음이온 그룹과의 전기적 중성화 등을 통해 세포의 항상성 유지, 팽압 조절, 세포 신장, 기공 개폐 과정들에서 중요한 역할을 수행한다 (Lebaudy et al. 2007; Gam-bale and Uozumi 2006). 이러한 K