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Ozone (O3), a potent air pollutant and a significant greenhouse gas, has been recognized as a major component of global climate change. However, current increasing trends in its background level are projecting a more severe threat to natural and cultivated plants in the near future. The present study has been designed to evaluate the impact of elevated concentrations of O3 on phenotypical, physiological, and biochemical traits in two high-yielding cultivars of wheat, followed by analysis of the leaf proteome using one/two-dimensional gel electrophoresis (1-/2-DGE) coupled to immunoblotting and mass spectrometry analyses under near-natural conditions using open top chambers. Prominently, O3 exposure caused specific foliar injury in both the wheat cultivars. Results also showed that O3 stress significantly decreased photosynthetic rate, stomatal conductance, and chlorophyll fluorescence kinetics (Fv/Fm) in test cultivars. Biochemical evaluations further revealed a higher loss in photosynthetic pigments, whereas a significantly induced antioxidant system under elevated O3 concentrations pointed toward an ability of O3 to generate oxidative stress. 1-DGE analysis showed drastic reductions in the abundantly present ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large and small subunits. Western blot analysis confirmed induced accumulation of antioxidative enzymes like superoxide dismutase and ascorbate peroxidase protein(s) and common defense/stress-related thaumatin-like protein(s). 2-DGE analysis revealed a total of 38 differentially expressed protein spots, common in both the wheat cultivars. Among those, some major leaf photosynthetic proteins (including RuBisCO and RuBisCO activase) and important energy metabolism proteins (including ATP synthase, aldolase, and phosphoglycerate kinase) were drastically reduced, whereas some stress/defense-related proteins (such as harpin-binding protein and germin-like protein) were induced. In all, the present study reveals an intimate molecular network provoked by O3 affecting photosynthesis and triggering antioxidative defense and stress-related proteins culminating in accelerated foliar injury in wheat plants.

Royal jelly (RJ) is an exclusive food for queen honey bee (Apis mellifera L.) that is synthesized and secreted by young worker bees. RJ is also widely used in medical products, cosmetics, and as health foods. However, little is known about RJ functionality and the total protein components, although recent research is attempting to unravel the RJ proteome. We have embarked on a detailed investigation of the RJ proteome, using a modified protein extraction protocol and two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with tandem mass spectrometry. Simultaneously, we examined total soluble protein from RJ collected at 24, 48, and 72 h after honey bee larvae deposition twice (in two flower blooming seasons), to check differences, if any, in RJ proteome therein. Both 1- and 2-D gels stained with silver nitrate revealed similar protein profiles among these three time points. However, we observed a clear difference in two bands (ca. MW of 55 and 75 kDa) on 1-D gel between the first and the second collection of RJ. A similar difference was also observed in the 2-D gel. Except for this difference, the protein profiles were similar at the 3 time points. As the RJ from 48 (or sometimes 72) is commercially used, we selected the RJ sample at 48 h for detailed analysis with the first collection. 1-DGE identified 90 and 15 proteins from the first and second selection, respectively; in total, 47 nonredundant proteins were identified. 2-DGE identified 105 proteins comprising 14 nonredundant proteins. In total, 52 nonredundant proteins were identified in this study, and other than the major royal jelly protein family and some other previously identified proteins, 42 novel proteins were identified. Furthermore, we also report potentially post-translationally modified (phosphorylation and glycosylation) RJ proteins based on the Pro-Q diamond/emerald phosphoprotein/glycoprotein gel stains; MRJP 2p and 7p were suggested as potential phosphoproteins. The 2-DGE data were integrated to develop a 2-D gel reference map, and all data are accessible through RJ proteomics portal (http://foodfunc.agr.ibaraki.ac.jp/RJP.html).

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.

Two global omics approaches were applied to develop an inventory of differentially expressed proteins and genes in Wig rat, a promising animal model of attention-deficit hyperactivity disorder (ADHD). The frontal cortex, striatum, and midbrain of Wig rat at 4 weeks of age were dissected for proteomics and transcriptomics analyses. Two-dimensional gel electrophoresis detected 13, 1, and 16 differentially expressed silver nitrate-stained spots in the frontal cortex, striatum, and midbrain, respectively. Peptide mass fingerprinting/tandem mass spectrometry identified 19 nonredundant proteins, belonging to 7 functional categories, namely, signal transduction, energy metabolism, cellular transport, protein with binding function, protein synthesis, cytoskeleton, and cell rescue. Interestingly, 10 proteins that were indentified in the present study were also previously reported in studies involving neurodegenerative diseases and psychiatric disorders, such as Alzheimer's disease (AD), Parkinson's disease, and Schizophrenia. Moreover, some of the proteins identified in the midbrain were involved in synaptic vesicular transport, suggesting abnormality in neurotransmitter release in this region. On the other hand, transcriptomics analysis of combined frontal cortex, striatum, and midbrain by rat whole genome 44K DNA oligo microarray revealed highly up-regulated (28) and down-regulated (33) genes. Functional categorization of these genes showed cellular transport, metabolism, protein fate, signal transduction, and transcription as the major categories, with 26% genes of unknown function. Some of the identified genes were related to AD, fragile X syndrome, and ADHD. This is a first comprehensive study providing insight into molecular components in Wig rat brain, and will help to elucidate the roles of identified proteins and genes in Wig rat brain, hopefully leading to uncovering the pathogenesis of ADHD.

Mushroom can be defined as a macrofungus with a distinctive fruiting body. Mushrooms of class Basidiomycete are primarily wood degradation fungi, but serve as food and a part of traditional medicine used by humans. Although their life cycle is fairly well-established, the information on the molecular components, especially proteins are very limited. Here, we report proteomics analysis of two edible mushrooms (fruiting bodies) Sparassis crispa and Hericium erinaceum using one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) based complementary proteomics approaches. 1-DGE coupled with liquid chromatography and mass spectrometry identified 77 (60 nonredundant proteins) and 121 (88 nonredundant proteins) proteins from S. crispa and H. erinaceum, respectively. 2-DGE analysis revealed 480 and 570 protein spots stained with colloidal coomassie brilliant blue in S. crispa and H. erinaceum, respectively. Of the 71 and 115 selected protein spots from S. crispa and H. erinaceum 2D gel blots on polyvinyldifluoride (PVDF) membranes, respectively, 29 and 35 nonredundant proteins were identified by N-terminal amino acid sequencing. Identified nonredundant proteins from 1- or 2-DGE belonged to 19 functional categories. Twenty-one proteins were found common in both S. crispa and H. erinaceum proteomes, including 14-3-3 protein and septin. Together this study provides evidence for the presence of a large number of functionally diverse proteins, expressed in the fruiting body of two economically important mushrooms, S. crispa and H. erinaceum. Data obtained from 1-DGE and 2-DGE analyses is accessible through the mushroom proteomics portal http://foodfunc.agr.ibaraki.ac.jp/mushprot.html.

Manduca sexta is an excellent insect model for studying insect physiology, including hemolymph proteins. Larvae stages of this insect are highly damaging to tobacco leaves causing a drastic decrease in crop yield. Investigation on the larval biology should help in controlling its destructive potential, thus increasing crop yields. The hemolymph is the source of its immunity to disease and environmental factors, which invariably involves protein components. To better understand the physiology of M. sexta and the protein components expressed during its life cycle, two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with N-terminal amino acid sequencing and liquid chromatography-mass spectrometry, were employed to analyze the fifth instar larvae hemolymph proteins. These proteomics approaches together identified 123 proteins, which constituted a total of 58 nonredundant proteins and belonged to 10 functional categories. Defense (49%), transport and metabolism (15%), storage (9%), and metamorphosis (7%) categories were highly represented accounting for 80% of the identified proteins. Besides identification of previously reported proteins, 18 novel proteins were identified, which include the lipoprotein-releasing system transmembrane protein lolC, 50S ribosomal protein L24, inducible serine protease inhibitor 1, imaginal disk growth factor, protein disulfide-isomerase-like protein ERp57, etc. The 2-DGE data were integrated to develop a 2-D gel reference map. Data obtained from 1-DGE and 2-DGE analyses are accessible through the M. sexta proteomics portal ( http://foodfunc.agr.ibaraki.ac.jp/mansehemoprot.html). Together, this study provides evidence for the presence of a large number of functionally diverse protein families in the hemolymph of M. sexta. These proteins correlate well with the fifth instar stage, the transition from larvae to pupae.

Unlabelled: Two global approaches were applied to develop an inventory of differentially expressed proteins and genes in rice (cv. Nipponbare) seedling grown on Murashige and Skoog medium with and without jasmonic acid (JA). JA significantly reduced the growth of shoot, root, leaf, and leaf sheath depending on JA concentration (1, 2, 5, 10, 25, and 50 microM) as compared with control. Almost 50% growth inhibition of seedling was observed with 5 microM JA. Shoots and roots of seedlings grown on 5 microM JA for 7 days were then used for proteomics and transcriptomics analyses. Two-dimensional gel electrophoresis revealed 66 and 68 differentially expressed protein spots in shoot and root, respectively, compared to their respective controls. Tandem mass spectrometry analysis of these proteins identified 52 (shoot) and 56 (root) nonredundant proteins, belonging to 10 functional categories. Proteins involved in photosynthesis (44%), cellular respiratory (11%), and protein modification and chaperone (11%) were highly represented in shoot, whereas antioxidant system (18%), cellular respiratory (17%), and defense-related proteins (15%) were highly represented in root. Transcriptomics analysis of shoot and root identified 107 and 325 induced genes and 34 and 213 suppressed genes in shoot and root, respectively. Except of unknown genes with over 57% of the total, most genes encode for proteins involved in secondary metabolism, energy production, protein modification and chaperone, transporters, and cytochrome P450. These identified proteins and genes have been discussed with respect to the JA-induced phenotype providing a new insight into the role of JA in rice seedling growth and development., Keywords: phytohormone * inhibitory concentration * growth * gel-based approach * mass spectrometry * DNA microarray.

Gamma knife surgery (GKS) is used for the treatment of various brain disorders. The biological effects of focal gamma ray irradiation on targeted or surrounding areas in the brain are not well-known. In the present study, we evaluated protein expression changes in the unilateral irradiated (60 Gy) striatum in rat. Striata of irradiated and control brains were dissected 16 h post-irradiation for analysis by large-format two-dimensional gel electrophoresis (2-DGE). In parallel, we also examined the un-targeted contralateral striatum over the control for potential changes in proteins patterns that may have occurred due to the effects of irradiation to the unilateral striatum. A total of 17 reproducible and differentially expressed silver nitrate-stained protein spots in the irradiated striatum was detected on 2-D gel. Their subsequent analysis by tandem mass spectrometry (nESI-LC-MS/MS) resulted in the identification of 13 nonredundant proteins. Interestingly, out of these 13 changed proteins, 2 proteins were also detected in the contralateral striatum. Some of the significantly changed proteins identified were creatine kinase, protein disulfide isomerase A3 precursor (PDA3), and peroxiredoxin 2 (Prx2). Western analysis with anti-PDA3 and anti-Prx2 antibodies revealed 4 and 2 cross-reacting protein spots on 2-D gel blots. Interestingly, after GKS, in the irradiated and un-irradiated striata, these spots showed a shift toward the acidic side, suggesting post-translational modifications. Taken together, these results indicate that unilateral irradiation during GKS triggers molecular changes in the bilateral striata.

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.