Understanding the host defense mechanisms in response to brown leaf spot disease caused by Cochliobolus miyabeanus is very important for production of resistant plant. In this study, two-dimensional gel electrophoresis (2-DGE) in conjunction with mass spectrometry was utilized to unravel changes of stress inducible proteins in rice leaves infected with C. miyabeanus. For this purpose, we firstly observed disease developmental process of C. miyabeanus in rice using trypan blue, anilin blue, acid fuchsin staining, and DAB staining for ROS detection and expressional abundance of ROS related proteins in rice leaves inoculated was confirmed by Western blotting. Proteins were extracted by PEG fractionation and their expression patterns were analyzed by 2-DGE and subjected to image analysis using the ImageMaster 6.0 2D Platinum software, resulting in the identification of 86 differentially expressed protein spots with significantly changes (p<0.05) compared with control. MALDI-TOFTOF-MS analysis revealed that 69 proteins including 42 and 27 significantly up- and down-regulated proteins, respectively, were identified. Based on gene ontology analysis, identified proteins were classified according to their functional groups: metabolism (20%), oxygen-detoxifying (13%), protein stress/defense (24%). Thus, these results for the first time suggest that differentially induced proteins may play a key role for understanding host defense mechanisms during rice -C. miyabeanus interaction.

The rice blast disease caused by Magnaporthe oryzae (M. oryzae) is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early responsible genes in rice in response to M. oryzae, we analyzed transcriptomics analysis using 300 K tilling microarray chip. The quality of RNA samples was initially validated by 4 defense related genes and phytoalexins measurement using RT-PCR and HPLC, respectively, which are well known defense markers. We determined that accumulation of 608 genes showed statistically significant changes in the level of transcription (>2 fold change, P<0.05). Among them, 261 genes were more up-regulated in incompatible interaction than that of compatible one. We further analyzed GO enrichment analysis of the 41 and 231 which were 2 fold up-regulated genes at 12h and 48h in incompatible interaction, respectively, using Rice Oligo nucleotide Array Database (http://ricearray.org). Furthermore, MapMan analysis (http://mapman.gabipd.org/) revealed that 21 and 85 genes including 18 receptor-like genes which were more induced in incompatible interaction compared to compatible interaction were found to be involved in biotic stress. Thus, this study suggests that early inducible genes including receptor-like protein kinases in incompatible interaction may play a key role in disease resistance against M. oryzae attacks.

Here, we first demonstrate that identification of rice brown spot disease fungus (Cochliobolus miyabeanus, C. miyabeanus) proteins is possible in infected tissues using in planta apoplastic proteome with non-destructive tissues. In planta apoplastic proteins from rice leaves inoculated with C. miyabeanus, CM2 (compatible race), were isolated by vacuum infiltration with CaCl2/Na-acetateextractionbuffer, separated by SDS-PAGE, and identified by MudPIT. Of the 529 proteins that were identified by MudPIT, a large proportion (490) was from the rice. Numerous carbohydrate metabolic process (48), oxidation and reduction (44), response to oxidative stress (20%) were identified and confirmed their expression at RNA levels using microarray. Bioinformatic analysis showed that 176 and 39 of these proteins have a signal peptide in rice and rice brown spot fungus, respectively, using Signal P. The large proportion of proteins interestingly identified from the in planta apoplast were involved inprotease, hydrophobin, and host cell wall hydrolysis (Xylanase, beta-glucosidase) derived from pathogen. Thus, we suggest that in planta rice apoplastic secretome will be an important clue to understand the rice-rice brown spot fungus interactions.

Development of transgenic plant with desirable traits to cultivated plant is one of the important procedures in plant molecular breeding. However, applicable assessment of transgenic plant in laboratorial scale is not much except cultivating transgenic plant for a whole life in field condition. Here, we analyzed chlorophyll fluorescence in three transgenic soybean lines with AtMYB44 transcription factor for assessment of photosynthetic activity under abiotic stresses such as drought. Soybean varieties used in this study were ‘Bert’ and ‘Bert’ derived three transgenic soybeans, ‘AtMYB44 CM35101’, ‘AtMYB44 CM2471’, and ‘AtMYB44 CM4481’. Analyzed five different chlorophyll fluorescence variables are maximum PSII quantum yield (QY_max), steady state PSII quantum yield (QY_Lss), steady state non-photochemical quenching (NPQ_Lss), coefficient of photochemical quenching in steady-state (Qp_Lss), and fluorescence declineratio in steady-state (Rfd_Lss). To determine main chlorophyll fluorescence variable affected by abiotic stress, principal component analysis (PCA) was conducted with five chlorophyll fluorescence variables measured from four varieties. QY_Lss and NPQ_Lss were main chlorophyll fluorescence variables to evaluate abiotic stress, particularly in drought stress. In comparison with transgenic soybean lines based on chlorophyll fluorescence variables, ‘AtMYB44 CM2471’ and ‘AtMYB44 CM4481’ are more tolerant to drought than the others. Interestingly, three transgenic soybean lines which have a same AtMYB44 gene with different regions of chromosome revealed the quite different responses of chlorophyll fluorescence to drought treatment.

Mesenchymal stem cells (MSC) are of great interest for cell-based therapies and tissue engineering approaches, as these cells are capable for extensive self-renewal and display a multilineage differentiation potential. Clinical application of these cells for degenerative and age-related diseases has been accumulating. However, preparation of MSC before the onset of the diseases, it needs to develop the cryopreservation method. Most cryopreservation methods include fetal bovine serum (FBS) which is essential for effective cryopreservation. Yet it should not be used clinically because of the potential risk of infection. In the present study, we investigated whether human serum albumin (HSA), human serum (HS), and knockout serum replacement (KSR) can be used as an alternative of FBS for cryopreservation of human adipose derived stem cells (hADSC). Cells cryopreserved with 9% HSA showed much higher viability after thawing compared with cells frozen with 5% or 1% HSA. Cells cryopreserved with 90% HS or KSR exhibited greater viability than cells frozen with 25% and 5% HS or KSR, respectively. Viability of cells frozen with 9% HSA, 90% HS or 90% KSR was comparable to that with 90% FBS. Morphology and proliferation ability of these cells were not affected by cryopreservation when compared the freshly obtained cells. Cryopreserved hADSC expressed transcription factor genes including Oct3/4, Nanog, Nestin and Sox2, which are related to the self-renewal of stem cells. Flow cytometric analyses showed that both fresh and cryopreserved hADSC were positive for the antigens of HLA-ABC, CD44, CD73, CD90, and CD105, CD166, and negative for HLA-DR, CD31, and CD34. Similar to fresh cells, cryopreserved hADSC could differentiate into mesodermal lineages, adipogenic, osteogenic, or chondrogenic cells. These results suggest that 9% HSA, 90% HS or 90% KSR can be used to replace FBS during successful cryopreservation of hADSC.

The human eyelid adipose-derived stem cells (HEACs) are known as a candidate source for stem cell-based therapy. HEACs possess the ability to proliferate in vitro and multipotency to differentiate into adipogenic, osteogenic and chondrogenic cells. To be used later than the time of collection, a long-term storage is needed. In this study, we investigated stem cell characteristics after cryopreservation of HEACs for 6 months and 1 year in liquid nitrogen. Frozen-thawed stem cells have shown that cumulative cell and doubling numbers were similar to those of fresh HEACs. After thawing, HEACs expressed stem cell-related genes of SCF, NANOG, OCT4, and TERT, ectoderm-related genes of NCAM and FGF5, mesoderm/endoderm-related genes of CK18 and VIM. They also consistently expressed transcripts of the immune-related genes of HLA-ABC and β2M. To induce mesodermal differentiation, cell were cultivated in adipogenic, osteogenic or chondrogenic medium for 2～3 weeks. After each differentiation culture, HEACs expressed adipocyte-, osteocyte- and chondrocytespecific genes. They were also stained with Oil red O, von Kossa, or alcian blue, revealing adipogenic, osteogenic, or chondrogenic character, respectively. The results suggest that long-term storage up to 1 year do not affect their biological properties, HEACs may be suitable for clinical application on cell-based therapies.