and distribution of seed storage proteins are responsible for the quality of soybean and seed development. Among storage proteins, lipoxygenase isoforms (Mw. ~97 kDa) play a major role in the distinct bean flavor during storage. In this study, we compared three soybean elite cultivars viz., JIMPUM, JINPUM2 and TANMI2 (lipoxygenase null mutants, originated from Japan) along with WILLIAMS 82 (control plant, USA) to determine the seed storage proteins by proteomic approach. Phenotype of the mature seeds showed the variation in seed coat, color and appearance. Total seed proteins of the above cultivars were subjected to two dimensional gel electrophoresis (2-DE). The resulted protein profiling showed the intensity of the different quantitative spots varied among the four cultivars. We are now investigating by using other proteomic tool and the resulted difference in proteins may helpful in quality improvement or the functional roles in the seed development.

Single seeds of common buckwheat cultivar Suwon No. 1 when subjected to SDS-PAGE revealed very high polymorphism. High variation existed for protein or protein subunits with molecular weight 54-47kDa, 45-25kDa and 16-11kDa. The electrophoregram showed variation for globulin as well as other protein fractions. About 300 proteins were separated by two-dimensional electrophoresis in common buckwheat (Fagopyrum esculentum Moench.) seed. Seed maturation is a dynamic and temporally regulated phase of seed development that determines the composition of storage proteins reserves in mature seeds. Buckwheat seeds from 5, 10, 15, 20, and 25 days after pollination and matured stage were used for the analysis. This led to the establishment of high-resolution proteome reference maps, expression profiles of 48 spots. It was identified 48 proteins from MALDI-TOF/MS analysis of wild buckwheat seed storage proteins. The 48 proteins were found identical or similar to those of proteins reported in buckwheat and other plants; it is belonging to 9 major functional categories including seed storage proteins, stress/defense response, protein synthesis, photosynthesis, allergy proteins, amino acid, enzyme, metabolism, and miscellaneous. It appears that the major allergenic storage protein separated played the important role in buckwheat breeding and biochemical characterization.

Buckwheat is one of the traditional crops and has become a renewed target of interest or a popular crop as a healthy foodstuff, because it is a good source of cereal protein which is rich with essential amino acids. However, what is critical to our health is that buckwheat contains proteins which cause a allergy. Buckwheat allergy resulting from ingestion is caused by the storage proteins in the grain with molecular weights ranging from 15, 22, 35, 39 and 50 kDa proteins of the inner fractions to low, and there were clear differences in the protein compositions between the inner and outer buckwheat flour fractions. A major allergenic protein of buckwheat is Fag e 1 with molecular weight 22 kDa (BW22KD). Buckwheat allergy is an immunoglobulin E (IgE)-mediated hypersensitive response capable of causing anaphylactic shock. Buckwheat seeds were dissected to endosperm and embryo. From each fraction we extracted proteins and analyzed extracts by SDS-PAGE and 2-DE. On electrophoregrams of endosperm proteins, 6 intense bands were detected. The most intense corresponded to molecular weights ranging from 54 to 65 kDa. These proteins have been reported not to be allergenic. We show here that the allergenic buckwheat seed proteins are found only among embryo proteins. No allergenic proteins were found in the buckwheat endosperm. The results presented here lead to the proposal that patients with hypersensitivity to buckwheat flour should use only fine flour from buckwheat endosperm, as this fraction contains no allergenic proteins. At present, specific protein spots will be selected and in-gel digested for MALDI-TOF-TOF/MS analysis.

We have discussed here the phenotypic and genetic characteristics as well as proteomic analysis of lesion mimic mutants (LLM) in rice. LLM is one of the mutants that induces cell death without infection of pathogen and produces defense signaling pathways. As the phenotypic expression, most LLMs induce spots on the leaf blades and leaf sheaths at their various developmental stages. We have discussed the nature of bl1, bl2, spl1, spl3, spl4, spl5 and spl6 LLMs in rice which were formed developmentally controlled spot on the leaf blades that were appeared as tiny dotted spots during tillering stage and gradually increased up to maturity. Through Northern blot analysis lower levels of rubisco large subunit and rubisco small subunit were observed in spotted leaves (sp) compared to non-spotted leaves (nsp). However, catalase was severely degraded in the sp. Broken thylakoid membranes of mesophyll chloroplasts were seen in nsp sections and were absent in sp sections of the mutant. Through 2-DE analysis 159 protein spots were differentially expressed between wild type and mutant from identified 800 reproducibleproteins, where 114 spots were up-regulated and 45 were down-regulated. Among quantified 25 protein spots, except two, all of the protein spots including protein disulfide isomerase, transketolase, thioredoxin peroxidase, ATP synthase, and rubisco large and small subunits were identified in the wild type but were absent in the mutant. However, catalase was up-regulated in the mutant. Genetic analysis indicated that studied bl1, bl2 and spl6 mutants are controlled by a single recessive gene.

The wheat-rye translocation lines have been agriculturally developed for the resistance to the biotypes of Hessian fly as a major insect pest of wheat. In order to compare the proteomic profiles between ‘Coker797’ (non-2RL), ‘Hamlet’ (2RL), and near-isogenic line (NIL) carrying 2RL, we evaluated the protein extraction and preparation methods for two-dimensional gel electrophoresis approach. The tissues such as leaves, stems, and roots from three wheat-rye lines were extracted by following trichloroacetic acid (TCA)/acetone precipitation. In a preliminary proteome analysis, a commonly expressed protein in Hamlet and NIL strain was identified as methionine synthase annotated in Hordeum vulgare subsp. The present study will provide the experimental guideline for the proteomic study of other useful crop plant tissues.

Plant proteomic study requires a high-throughput separation to the detection and analysis of peptides and proteins by mass spectrometry (MS) to detect low abundant proteins. Together, efficient separation and MS can lead to the detection of thousands of plant proteins in a cell or tissue and help build proteome maps that can provide a global snap-shot of the cell or tissue status. Recently efficient separations based on the HPLC were introduced to allow deeper protein detection and improve throughput. For the HPLC based methods, Multidimensional Protein Identification Technology (MudPIT) and 1D-Gel-LC-MS/MS will be introduced. In MudPIT analysis, all proteins in a sample are digested into peptides before the separation step then the mixture of peptides are separated through the biphasic capillary column and sequentially eluted into the mass spectrometer and analyzed. 1D-Gel-LC-MS/MS separates protein samples in 1D-SDS-Gel then the proteins in each band were ingel-digested into peptides followed by peptides separation with Reverse Phase column and elution into the mass spectrometer. The main goal of this presentation is to introduce the recent protein separation and identification methods based on the HPLC coupled with MS analysis including conventional method of 2D-PAGE.

태양광과 반응하여 독특한 광화학적 작용을 하는 이산화티탄(TiO2 )을 벼 잎 표면에 처리하였을 때 벼 엽신의 광합성 대사에 대한 영향을 검토하고 프로테옴 분석을 통해 생리변화를 구명하고자 수행한 결과를 요약하면 다음과 같다. 1. 광합성유효파장이 2,400umolm2s1과 2,200umolm2s1 배치구에서 이산화티탄 10, 20 ppm 처리는 광적응상태의 엽록소형광지수(Yield)를 낮추었고 450umolm2s1 처리구는 엽록소형광지수를 높였다. 2. 노지조건인 PAR 2,400umolm2s1 배치구에서 광합성 명반응의 상대전자전달율은 이산화티탄 10 ppm 처리에서 평균 45 %, 무처리 32.4 %, diuron 10 ppm 처리구에서 15.3%로 이산화티탄 처리는 광합성 명반응의 상대전자전달율을 높였다. 3. UV-B 4.9, 0.6KJm2day1 배치구에서 이산화티탄 처리로 초장이 증가하였고 UV-B 0.15KJm2day1 배치구에서 초장은 증가하고 건물중은 감소하였다. 4. 광합성은 노지의 UV-B 조건인 13.6KJm2day1 배치구에서 이산화티탄 처리로 종가하였고 UV-B 4.9, 0.6, 0.15KJm2day1 배치구는 다소 증가하였으나 통계적으로 유의한 차이는 나타내지 않았다. 5. 이산화티탄 처리 후 자연광 중의 UV-B를 99% 차단하여 저수준으로 조절한 결과 68%의 단백질 발현이 감소하였고 각각 16%의 단백질 발현이 증가 또는 신생 합성되었다. 6. 이산화티탄 20 ppm 처리 후 자연광 중의 UV-B를 99% 차단시켰을 때 주로 광합성 Calvin cycle에서 CO2 결합을 촉매하는 결정구조 Rubisco의 chain E 발현이 감소하였다.