Two-dimensional electrophoresis (2-DE) was executed to separate the seed storage proteins from the buckwheat. The proteins extracted from the whole seed proteins were better separated and observed in the use of lysis buffer. Using this method, the highly reproducible isoelectric focusing (IEF) can be obtained from polyacrylamide gels, and IEF from the polyacrylamide gel at all the possible pH range (5.0-8.0) was more easily separated than IPG (immobilized pH gradient) gels. The polyacrylamide gels in the first dimension in 2-DE was used to separate and identify a number of whole seed proteins in the proteome analysis. In this new apparatus using 2-DE, 27cm in length of plate coated with polyacrylamide gel was used and the experiment was further investigated under the various conditions.

Salt stress is one of the major abiotic stresses affecting the yield of ginseng (Panax ginseng C. A. Meyer). The objective of this study was to identify bio-marker, which is early responsive in salt stress in ginseng, using proteomics approach. Ginseng plants were exposed to 5 ds/m salt concentration and samples were harvested at 0, 6, 12 and 18 hours after exposure. Total proteins were extracted from ginseng leaves treated with salt stress using Mg/NP-40 buffer and were separated on high resolution 2-DE. Approximately 1003±240 (0 h), 992±166 (6 h), 1051±51 (12 h) and 990±160 (18 h) spots were detected in colloidal CBB stained 2D maps. Among these, 8 spots were differentially expressed and were identified by using MALDI-TOF/TOF MS or/and LC-MS/MS. Ethylene response sensor-1 (spot GL 1), nucleotide binding protein (spot GL 2), carbonic anhydrase-1 (spot GL 3), thylakoid lumenal 17.9 kDa protein (spot GL 4) and Chlorophyll a/b binding protein (spot GL 5, GL 6) were up-regulated at the 12 and 18 hour, while RuBisCO activase B (spot GL 7) and DNA helicase (spot GL 8) were down-regulated. Thus, we suggest that these proteins might participate in the early response to salt stress in ginseng leaves.

Soybean seed is a good source of plant protein in human consumables such as baby formula and protein concentrate. The seeds contain an abundance of storage proteins, namely β-conglycin and glycinin that account for ~ 70-80% of the total seed protein content. Proteome profiling has been proved to be an efficient way that can help us to investigate the seed storage proteins. In the present study, the seeds were removed from the pods and the cotylendonary tissues were separated from the testa for proteome analysis in order to investigate the seed storage proteins. A systematic proteome profiling was conducted through one-dimensional gel electrophoresis followed by MALDI-TOF-TOF mass spectrometry in the seeds (cotyledonary tissue) of soybean genotypes. Two dimensional gels stained with CBB, a total of 10 proteins were identified and analyzed using MASCOT search engine according to the similarity of sequences with previously characterized proteins along with the UniProt database. A total of ten proteins such as glycinin Gy4 precursor, glycinin G3 precursor, glycinin G1 precursor, glycinin chain A2B1a precursor, glycinin chain A2B1a precursor were identified in our investigation. However, the glycinin subunit may be considered to play important roles in soybean breeding and biochemical characterization. In addition, the improved technique will be useful to dissect the genetic control of glycinin expression in soybean.

The aim of this study was to analyze the effect of antifreeze proteins (AFPs) on vitrification of mouse mature (MII) oocytes. We studied about 3 types of AFPs from different origins (FfIBP, LeIBPand Type III AFP). The MII oocytes were obtained from 4-week-old BD-F1 mice. Vitrification of oocyte was performed by 2 steps using the Cryotop (equilibration: 7.5% EG + 7.5% PROH for 5 min, vitrification: 15% EG + 15% PROH + 0.5M sucrose for 1 min). The concentrations of AFPs added to these solutions were 0.05 mg/ml for FfIBP and 0.1 mg/ml for LeIBP and Type III AFP. After fertilization, embryo development was assessed up to 5 days. Through immunostaining of vitrified-warmed oocytes, we assessed the normal meiotic spindle. Also, intracellular ROS and mitochondrial activity was analyzed. In the developmental stages, FfIBP and LeIBP groups showed significantly higher survival rates. In the blastocyst and apoptotic blastomere rates were significant differences in AFPs treated groups. AFPs treated groups were significantly higher in blastocyst cell numbers than control group. Among the AFPs treated groups, FfIBP, LeIBP groups were significantly higher rates. And, in cleavage rates, FfIBP group was significantly higher rates than the other groups. In vitrified-warmed MII oocytes, the normal meiotic spindle organization and chromosome alignment rate was significantly higher in FfIBP and LeIBP groups. And in intracellular ROS levels, control group was significantly increased than AFPs treated groups. However, in the mitochondrial activity, LeIBP group was significantly higher than control, FfIBP and LeIBP groups. AFPs treated groups were significant differences in development, meiotic spindle organization and intracellular ROS levels. And in the AFPs treated groups, FfIBP and LeIBP groups were significantly higher rates in normal meiotic spindle and mitochondrial activity than Type III AFP group respectively. In conclusion, FfIBP and LeIBP can be thought to improve oocyte cryopreservation efficiency.

As the drought is getting worse, Lot of studies related to drought stress in plant have been conducted. Recently whole genome sequencing of Brassica rapa ssp which is important vegetable crop to East Asians has been completed to enable Omic research. It is known that the drought damages occur in the early stage of plant development. Here, we performed shotgun proteomics analysis of B. rapa to observe the morphological characters, monitor the expression patterns of the identified proteins during drought stress, and detect the proteins related to drought stress. The three week old B. rapa grown in density of single plant in a single pot were used. Drought stress were treated as that a single plant in soil was removed from the pot and the plant with soil was exposed to air and light without watering. Leaves were immediately harvested before drought treatment, 24hr after drought treatment, and 48hr after drought treatment. The protein expression patterns were monitored by a quantitative shotgun proteomics analysis. Extracted proteins were separated in 1D-SDS-PAGE then the gel sliced into seven pieces. Chopped gels were ingel-digested. Peptides were assigned to mass spectrometry (Q-Exactive). The ms/ms spectra were analyzed through Proteome Discoverer. By combining all of the identified proteins in the seven sliced gel samples, total B. rapa proteome reference map was completed. Protein expression patterns were investigated by comparing the quantity of protein. With shotgun proteomic approach, we evaluated the changes in the quantity and finally discovered the candidate proteins related with drought stress.

Arabidopsis atDjC53 and atDjC32 gene DnaJ-like protein homologous to DnaJ-like protein was characterized for the functional analysis of DnaJ-like protein. It was shown that atDjC53 and atDjC32 RNA expression is induced by heat shock stress and atDjC53- and atDjC32-GFP was targeted to the nucleus of protoplasts. The atDjC53 and atDjC32 promoter (1 kb) was isolated and fused to the GUS reporter gene to investigate gene regulation of atDjC53 and atDjC32 specific to heat shock stress or to developmental organ in the transgenic lines. RNAi and overexpression construct was employed to generate atDjC53 and atDjC32 knock-out plants for the study of their function. Molecular function of atDjC53 and atDjC32 is discussed in relation to heat shock and also developmental stages in Arabidopsis.

High temperature impediment in developing stages of crops has been occurred due to the impact of global warming. Rice production is notable to be sensitive to increasing environmental temperature and grain filling temperatures are already approaching threatening levels in many countries with rice cultivation. Recent proteomic analyses exposed impulsive changes of metabolisms during rice grain development. Interestingly, proteins involved in glycolysis, citric acid cycle, lipid metabolism, and proteolysis were accumulated at higher levels in mature grain than those of developing stages. High temperature (HT) stress in rice ripening period enhances damaged (chalky) grains which have loosely compacted shape starch granules. We carried out two-dimensional gel electrophoresis to analyze protein profiles during grain filling and different developmental stages of rice seed maturation. Proteins were separated from the fertilized seeds (seeds from 7 days and 21 days after fertilization) and seed maturation stage using IEF in the first-dimension and SDS-PAGE in the second dimension along with MALDI-TOF mass spectrometry. More than 1,000 protein spots were detected on a two-dimensional gel electrophoresis. A total of 120 different protein spots out of 140 protein spots were identified by MALDI-TOF and nano LCQ-TOF mass spectrometer. The identified proteins were categorized into six (6) different groups according to their expression patterns during grain filling and seed maturation. Some proteins were confirmed during seed development stages such as cytoplasmic malate dehydrogenase, whereas others were appeared at a specific stage like putative subtilisin-like protease, germin-like, seed allergenic proteins. Furthermore, the chalking mechanism of rice grain under the HT stress could be discussed in terms of grain starch glycome, transcriptome, and proteome.

Chilling stress affects growth and yield of warm-climate crops such as soybean (Glycine max L.) that is susceptible to low temperature (10-18℃). A comparative proteomic approach was employed to explore the mechanisms underlying soybean response to chilling stress. Soybean seedlings were germinated for 3-4 days and exposed to low temperature (10℃) for 3 days, and the proteins were extracted from seedling leaves. Protein separation by SDS-PAGE followed by liquid chromatography electro-spray ionization tandem mass spectrometry (LC-ESI MS/MS) was effective approach to identify proteins, based on the number of peptides reliably identified. A total of 77 proteins out of 704 proteins were identified in the presence of chilling stress. Most proteins identified had functions related to cell signaling, metabolism, energy and transport, protein biosynthesis and degradation, cytoskeleton, and were involved in regulating reactions and defending against stress. It is therefore likely that the response of soybean plant’s proteome to chilling stress is complex, and that the identification proteins may play an important role in regulating adaptation activities following challenge to chilling stress to facilitate cellular homeostasis. Furthermore, our result suggest that new ways of engineering stress-tolerant plants responding climate change by providing outline for agriculturally important chilling stress.

Arabidopsis atDjC53 and atDjC32 gene DnaJ-like protein homologous to DnaJ-like protein was characterized for the functional analysis of DnaJ-like protein. It was shown that atDjC53 and atDjC32 RNA expression is induced by heat shock stress and atDjC53- and atDjC32-GFP was targeted to the nucleus of protoplasts. The atDjC53 and atDjC32 promoter (1 kb) was isolated and fused to the GUS reporter gene to investigate gene regulation of atDjC53 and atDjC32 specific to heat shock stress or to developmental organ in the transgenic lines. RNAi and overexpression construct was employed to generate atDjC53 and atDjC32 knock-out plants for the study of their function. Molecular function of atDjC53 and atDjC32 is discussed in relation to heat shock and also developmental stages in Arabidopsis.

Rice seed storage proteins (SSPs) are accumulated in storage organelles of the endosperm during seed maturation. The SSPs from the rice seeds consist of glutelins as a major SSP, and prolamins and globulins comprise about the rest 20 % of the SSPs. To improve the nutritional quality of rice seeds or processing properties of rice flour, we are attempting to change the composition of the SSPs in rice seeds. For this purpose, we generated many transgenic rice plants, which show the altered levels of the SSPs, by using the RNA interference (RNAi). Accumulation of glutelins was 76% reduced in the GluA-RNAi lines. The Pro-RNAi lines revealed the reduced levels of prolamins to 36%. The protein level of globulins was 61% reduced in the Glb-RNAi lines. Interestingly, an obvious reduction of glutelins, prolamins, and globulins was not examined in the GluA:Pro:Glb-RNAi lines. This suggests that a reduction of a few SSPs could be compensated by the increases of other SSPs at the protein levels. We are also attempting to generate transgenic rice plants expressing both a high-molecular-weight (HMW) glutelin subunit and a low-molecular-weight (LMW) glutelin subunit. These manipulations of rice SSPs might be an important contribution on improving the functional properties of rice seeds.

Recently, proteome analysis is becoming a powerful tool for the functional characterization of plants. Due to the availability of vast nucleotide sequence information and based on the progress achieved in sensitive and rapid protein identification by mass spectrometry, proteome approaches open up now perspectives to analyze the complex functions of model crop species at different level. In this study, we have N-terminal sequencing data for the 100 embryo and 53 seed proteins of rice separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) were collected and systematically organized for a protein sequence data-file. An attempt was made to link the embryo proteins of rice to DNA sequences for understanding their functions. One hundred proteins of the 700 spots were detected in the embryo using 2-DE gels whereas we used micro sequenced. Of these, 28% of the embryo proteins were matched to DNA sequences with known functions, but 72% of the proteins were identified to be unknown functions as previously reported by Woo et al.,. In addition, twenty-four spots of protein with 100% of homology and nine with over 80% were matched to ESTs (expressed sequence tags) after expanding the amino acid sequences of the protein spots by Database searches using the available EST databases of rice at the NCBI (http://www/ncbi.nlm.nih.gov/) and DDBJ (http://www.ddbj.nig.ac.jp/). Also, a total of 53 proteins out of 700 protein spots separated on the 2-DE gels were analyzed by the peptide mass fingerprinting method (MALDI-TOF/MS). High-quality mass spectra suitable for peptide mass fingerprinting were obtained from 41 spots. Using the ESI-Q-TOF/MS, however, we were able to identify 53 seed proteins of rice, including 12 proteins not registered in database. The rapid expansion of DNA sequence databases to the utilization of EST now provides the whole or partial gene sequences of model organisms, and the recent advances in protein micro-characterization by mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. Proteome Database of rice is updated, and is available on the World Wide Web at http://gene64.rda.affrc.go. This work shows that the proteome analysis could be a useful strategy to link the sequence information to the functional genomics.

We improved the separation of the basic proteins from the soybean cotyledon, Glycine max L. Merr. by searching N-terminal sequences data in proteins isolated by two-dimensional electrophoresis (2-DE). After removed Hexane, proteins were extracted from cotyledon with a urea/Triton/2-mercaptoetanol solution. Using this method, the highly reproducible isoelectric focusing (IEF) can formed with polyacrylamide gels with pH 4.0-9.8. The IEF tube gels were used as the first dimension, and proteins were visualized by second-dimensional gel electrophoresis, and identify a number of soybean cotyledon proteins using mass spectrometry in the proteome analysis. These instruments of 2-DE and IEF tube gels were used 27 cm and investigate under various conditions. The total number of spots and features was obtained by PDQuest software (Bio-Rad). In this experiments performed, the IEF tube gels and instruments afforded good reproducibility in the number of PDQuest-detected spots from gel to gel while IPG offered better reproducibility in the total number of manually detected spots from gel to gel. In conclusion, we have separated of the basic 13 proteins in soybean. The glycinin subunit separations are also considered to play important roles in soybean breeding and biochemical characterization. The improved technique will be useful to dissect the genetic control of glycinin expression in soybean.