By using an in vivo hydroponic rice seedling culture system, we investigated the physiological and biochemical responses of a model rice japonica cultivar Nipponbare to salt stress using proteomics and classical biochemical methods. Yoshida's nutrient solution (YS) was used to grow rice seedlings. YS-grown 18-day-old seedlings manifested highly stable and reproducible symptoms, prominently the wilting and browning of the 3rd leaf, reduced photosynthetic activity, inhibition in overall seedling growth, and failure to develop new (5th) leaf, when subjected to salt stress by transferring them to YS containing 130 mM NaCl for 4 days. As leaf response to salt stress is least investigated in rice by proteomics, we used the 3rd leaf as source material. A comparison of 2-DE protein profiles between the untreated control and salt-stressed 3rd leaves revealed 55 differentially expressed CBB-stained spots, where 47 spots were increased over the control. Of these changed spots, the identity of 33 protein spots (27 increased and 5 decreased) was determined by nESI-LC-MS/MS. Most of these identified proteins belonged to major metabolic processes like photosynthetic carbon dioxide assimilation and photorespiration, suggesting a good correlation between salt stress-responsive proteins and leaf morphology. Moreover, 2-DE immunoblot and enzymatic activity analyses of 3rd leaves revealed remarkable changes in the key marker enzymes associated with oxidative damage to salt stress: ascorbate peroxidase and lipid peroxidation were induced, and catalase was suppressed. These results demonstrate that hydroponic culture system is best suited for proteomics of salt stress in rice seedling.

Ozone (O(3)), a serious air pollutant, is known to significantly reduce photosynthesis, growth, and yield and to cause foliar injury and senescence. Here, integrated transcriptomics, proteomics, and metabolomics approaches were applied to investigate the molecular responses of O(3) in the leaves of 2-week-old rice (cv. Nipponbare) seedlings exposed to 0.2 ppm O(3) for a period of 24 h. On the basis of the morphological alteration of O(3)-exposed rice leaves, transcript profiling of rice genes was performed in leaves exposed for 1, 12, and 24 h using rice DNA microarray chip. A total of 1535 nonredundant genes showed altered expression of more than 5-fold over the control, representing 8 main functional categories. Genes involved in information storage and processing (10%) and cellular processing and signaling categories (24%) were highly represented within 1 h of O(3) treatment; transcriptional factor and signal transduction, respectively, were the main subcategories. Genes categorized into information storage and processing (17, 23%), cellular processing and signaling (20, 16%) and metabolism (18, 19%) were mainly regulated at 12 and 24 h; their main subcategories were ribosomal protein, post-translational modification, and signal transduction and secondary metabolites biosynthesis, respectively. Two-dimensional gel electrophoresis-based proteomics analyses in combination with tandem mass spectrometer identified 23 differentially expressed protein spots (21 nonredundant proteins) in leaves exposed to O(3) for 24 h compared to respective control. Identified proteins were found to be involved in cellular processing and signaling (32%), photosynthesis (19%), and defense (14%). Capillary electrophoresis-mass spectrometry-based metabolomic profiling revealed accumulation of amino acids, gamma-aminobutyric acid, and glutathione in O(3) exposed leaves until 24 h over control. This systematic survey showed that O(3) triggers a chain reaction of altered gene, protein and metabolite expressions involved in multiple cellular processes in rice.

Secreted proteins control a multitude of biological and physiological processes in multicellular organisms such as plants. Identification of secreted proteins in reference plants like Arabidopsis and rice under normal growth conditions and adverse environmental conditions will help better understand the secretory pathways. Here, we have performed a systematic in planta and in vitro analyses of proteins secreted by rice leaves (in planta) and seed callus suspension-cultured cells (SCCs; in vitro), respectively, using a combination of biochemical and two-dimensional gel electrophoresis (2-DGE) coupled with liquid chromatography mass spectrometry analyses. Secreted proteins prepared from either leaves or SCCs medium were essentially free from contamination of intracellular proteins as judged by biochemical and Western blot analyses. 2-DGE analyses of secreted proteins collectively identified 222 protein spots with only 6 protein spots common to both in planta and in vitro derived data sets. Data were used to establish high-resolution and high-density 2-D gel reference maps for both in planta and in vitro secreted proteins. Identified proteins belonged to 11 (in planta) and 6 (in vitro) functional classes. Proteins involved in carbon metabolism (33%) and cell wall metabolism having plant defense mechanism (18%) were highly represented in the in planta secreted proteins accounting for 51% of total identified proteins, whereas proteins of cell wall metabolism having plant defense mechanism (64%) were predominant in the in vitro secreted proteins. Interestingly, secreted proteins possessing signal peptides were significantly lower in an in planta (27%) prepared secreted protein population than in vitro (76%) as predicted by SignalP prediction tool, implying the notion that plant might possess yet unidentified secretory pathway(s) in addition to the classical endoplasmic reticulum/Golgi pathway. Taken together, this systematic study provides evidence for (i) significant difference in protein population secreted in planta and in vitro suggesting both approaches are complementary, (ii) identification of many novel and previously known secreted proteins, and (iii) the presence of large number of functionally diverse proteins secreted in planta and in vitro.

Background: The 2015 MERS outbreak in South Korea was the largest event outside of the Middle East. Under such circumstances, individuals tend to resort to non-conventional solutions such as complementary and alternative medicine (CAM) to manage health. Thus, this study aims to examine characteristics of CAM use among outpatients in a community hospital setting during the 2015 MERS outbreak and to assess potential predictors of CAM use during the epidemic. Methods:A cross-sectional study was conducted among 331 patients (response rate: 82.75%) at a community hospital located in Seoul, South Korea. The survey instrument included 36 questions on the use of CAM, demographic characteristics, health status, and respondents' perceptions and concerns about MERS infection. Chisquare test and logistic regression were conducted for data analysis using SPSS ver. 21.0., and a p-value of less than 0.05 was considered statistically significant for all analyses. Results: 76.1% of respondents used one or more types of CAM modalities during the MERS outbreak. Consumption of easily accessible modalities such as multivitamin (51.2%) and food products (32.1%) was most popular, and the majority of CAM users relied on mass media (52.4%) and the internet (27.4%) to obtain information on CAM. The use of CAM was associated with age between 40 and 49, age over 50, prior CAM use, and dissatisfaction with the government response to the MERS outbreak.Conclusions: CAM was commonly used by outpatients during the 2015 MERS outbreak in Korea, and mass media was the main source of information. Establishing a media platform is of paramount importance to provide reliable information and ensure the safety of its use.

A metabolomics based approach has been used to study the infection of the Hwacheong rice cultivar (Oryza sativa L. cv. Hwacheong) with compatible (KJ201) and incompatible (KJ401) strains of the rice blast fungal pathogen Magnaporthe grisea. The metabolic response of the rice plants to each strain was assessed 0, 6, 12, 24, 36, and 48 h post inoculation. Nuclear Magnetic Resonance (NMR) spectroscopy and Gas and Liquid Chromatography Tandem Mass spectrometry (GC/LC-MS/MS) were used to study both aqueous and organic phase metabolites, collectively resulting in the identification of 93 compounds. Clear metabolic profiles were observed at each time point but there were no significant differences in the metabolic response elicited by each pathogen strain until 24 h post inoculation. The largest change was found to be in alanine, which was~30% (±9%) higher in the leaves from the compatible, compared to the resistant, plants. Together with several other metabolites (malate, Eur J Plant Pathol (glutamine, proline, cinnamate and an unknown sugar) alanine exhibited a good correlation between time of fungal penetration into the leaf and the divergence of metabolite profiles in each interaction. The results indicate both that a wide range of metabolites can be identified in rice leaves and that metabolomics has potential for the study of biochemical changes in plant-pathogen interactions.

Rice Oryza sativa accelerated cell death and resistance 1 (OsACDR1) encodes a putative Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK). We had previously reported upregulation of the OsACDR1 transcript by a range of environmental stimuli involved in eliciting defense-related pathways. Here we apply biochemical, gain and loss-of-function approaches to characterize OsACDR1 function in rice. The OsACDR1 protein showed autophosphorylation and possessed kinase activity. Rice plants overexpressing OsACDR1 exhibited spontaneous hypersensitive response (HR)-like lesions on leaves, upregulation of defense-related marker genes and accumulation of phenolic compounds and secondary metabolites (phytoalexins). These transgenic plants also acquired enhanced resistance to a fungal pathogen (Magnaporthe grisea) and showed inhibition of appressorial penetration on the leaf surface. In contrast, loss-offunction and RNA silenced OsACDR1 rice mutant plants showed downregulation of defense-related marker genes expressions and susceptibility to M. grisea. Furthermore, transient expression of an OsACDR1:GFP fusion protein in rice protoplast and onion epidermal cells revealed its localization to the nucleus. These results indicate that OsACDR1 plays an important role in the positive regulation of disease resistance in rice.

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We analyzed a unique rice (Oryza sativa L.) blast lesion mimic (blm) mutant for differentially expressed proteins in leaves of one- and two-week-old seedlings manifesting the lesion mimic phenotype. Gel-based one- and two-dimensional electrophoresis (1- and 2-DGE) was performed using leaves (blm and wild-type, WT) before (stage 1, S1) and after (stage 2, S2) lesion formation. 1-DGE immunoblotting revealed potent increase in the expression of a key pathogenesis-related (PR) marker biosynthetic enzyme, naringenin 7-O-methyltransferase, involved in rice phytoalexin sakuranetin biosynthesis, and three oxidative-stress-related marker proteins, catalase, ascorbate peroxidase (APX), and superoxide dismutase (SOD) in leaves of the blm mutant. 2-D gel immunoblotting analysis with anti-APX and anti-SOD antibodies revealed newly appearing cross-reacting protein spots in blm. 2-DGE analysis detected 50 Coomassie brilliant blue-stained protein spots differentially expressed in blm. A total of 23 and 44 protein spots was excised for analysis by N-terminal amino acid sequencing and nano-electrospray ionization liquid chromatography mass spectrometry, respectively; 26 nonredundant proteins were identified. The pathogenesis-related class 5 and 10 proteins, including a new OsPR10d protein, were significantly induced in blm. The OsPR5 protein spot was stained with Pro-Q Diamond phosphoprotein gel stain suggesting OsPR5 to be a putative phosphoprotein. Surprisingly, protein spot 20, a leaf OsPR10b, showed identity to a rice root-specific PR-10 (RSOsPR10). To resolve this discrepancy, we checked its expression in leaves of blm and WT (S1 and S2), respectively, using gene-specific primers and reverse transcriptase-polymerase chain reaction; RSOsPR10 mRNA was found to express in the leaves.