Proteomics Identification of Differentially Expressed Proteins Relevant for Nicotine Synthesis in Flue-Cured Tobacco Roots Before and After Decapitation

Nicotine is a secondary substance synthesized in tobacco roots. In flue-cured tobacco planting, tobacco decapitation is an effective practice to promote nicotine biosynthesis by regulation of the redistribution of total nitrogen amounts. However, proteins relevant to nicotine synthesis in tobacco roots has not been identified and characterized yet. It is important to explore the regulation of nicotine biosynthesis in tobacco roots. To identify the proteins relevant to nicotine synthesis, the protein patterns in roots of flue-cured tobacco （cv. K326） before and after decapitation were analyzed. In the present study, the protein patterns in roots of flue-cured tobacco were analyzed by two-dimensional electrophoresis （2-DE）, and the differentially-expressed spots were identified by matrix assisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF-MS）. Paired comparison of 2-DE maps revealed 26 spots of differentially-expressed proteins in roots before and after decapitation. Furthermore, nine differentially-expressed spots were identified. There were four proteins which were enzymes possibly involved in nicotine biosynthesis. In addition, the roles of the four enzymes in nicotine biosynthesis were discussed in a putative network. Our results would contribute to the understanding of the regulation pathway of nicotine biosynthesis and further to the molecular manipulation on the nicotine contents in flue-cured tobacco.

Proteomics Identification of Differentially Expressed Leaf Proteins in Response to Setosphaeria turcica Infection in Resistant Maize

Northern corn leaf blight （NCLB）, caused by the heterothallic ascomycete fungus Setosphaeria turcica, is a destructive foliar disease of maize and represents a serious threat to maize production worldwide. A comparative proteomic study was conducted to explore the molecular mechanisms underlying the defense responses of the maize resistant line A619 Ht2 to S. turcica race 13. Leaf proteins were extracted from mock and S. turcica-infected leaves after inoculated for 72 h and analyzed for differentially expressed proteins using two-dimensional electrophoresis and mass spectrometry identification. 137 proteins showed reproducible differences in abundance by more than 2-fold at least, including 50 up-regulated proteins and 87 down-regulated proteins. 48 protein spots were successfully identified by MS analysis, which included 10 unique, 6 up-regulated, 20 down-regulated and 12 disappeared protein spots. These identified proteins were classified into 9 functional groups and involved in multiple functions, particularly in energy metabolism （46%）, protein destination and storage （12%）, and disease defense （18%）. Some defense-related proteins were upregulated such as 13-glucosidase, SOD, polyamines oxidase, HSC 70 and PPIases; while the expressions of photosynthesis- and metabolism-related proteins were down-regulated, by inoculation with S. turcica. The results indicated that a complex regulatory network was functioned in interaction between the resistant line A619 Ht2 and S. turcica. The resistance processes of A619 Ht2 mainly resided on directly releasing defense proteins, modulation of primary metabolism, affecting photosyntesis and carbohydrate metabolism.

Differential Proteomic Analysis of Leaf Development at Rice (Oryza sativa) Seedling Stage

To identify the function of differential expression proteins in different leaves of rice seedlings extracted from 2- to 5-leaf stages, the leaf proteins at the seedling stage of hybrid rice Shanyou 63 were studied by using the approach of plant proteomics, and those proteins were separated with two-dimensional electrophoresis （2-DE） and then analyzed with an imagemaster 2D Elite 5.0. The results showed that the 41 protein spots were detected differential expression, of which 17 new protein spots appeared after the 3-leaf stage, including 9 special protein spots, which were only detected at the 3-leaf stage. Thirteen protein spots increased first and then decreased in expression abundance gradually and finally even disappeared. For the other 11 protein spots, 3 protein spots decreased, but 6 protein spots were opposite in expression abundance, however, 2 protein spots expressed in an irregular pattern after the 2-leaf stage. Of the 41 differential leaf proteins, 15 protein spots were identified by ESI-Q MS/MS and categorized into 4 groups of functions. The results indicated that proteins were the carriers of the functions in cells, but were significantly influenced by the changes in cell function or intercellular environment; hence, the reason that caused the proteomic changes as mentioned earlier might be related to the occurrence of tillers at the rice seedling stage after the 3-leaf stage.

Differential Proteomics Reveals the Potential Injury Mechanism Induced by Heavy Ion Radiation in Mice Ovaries

In the present study, we used a proteomics approach based on a two-dimensional electrophoresis （2-DE） reference map to investigate protein expression in the ovarian tissues of pubertal Swiss-Webster mice subjected to carbon ion radiation （CIR）. Among the identified proteins, ubiquitin carboxy-terminal hydrolase L1 （UCH-L1） is associated with the cell cycle[1] and that it influences proliferation in ovarian tissues. We analyzed the expression of UCH-L1 and the proliferation marker proliferation cell nuclear antigen （PCNA） following CIR using immunoblotting and immunofluorescence. The proteomics and biochemical results provide insight into the underlying mechanisms of CIR toxicity in ovarian tissues.

Potential targets for protecting against hippocampal cell apoptosis after transient cerebral ischemiareperfusion injury in aged rats

Mitochondria play an important role in neuronal apoptosis caused by cerebral ischemia, and the role is mediated by the expression of mitochondrial proteins. This study investigated the involvement of mitochondrial proteins in hippocampal cell apoptosis after transient cerebral ischemia-reperfusion injury in aged rats using a comparative proteomics strategy. Our exper-imental results show that the aged rat brain is sensitive to ischemia-reperfusion injury and that transient ischemia led to cell apoptosis in the hippocampus and changes in memory and cognition of aged rats. Differential proteomics analysis suggested that this phenomenon may be mediated by mitochondrial proteins associated with energy metabolism and apoptosis in aged rats. This study provides potential drug targets for the treatment of transient cerebral isch-emia-reperfusion injury.

Electroacupuncture pretreatment exhibits antidepressive effects by regulating hippocampal proteomics in rats with chronic restraint stress

The clinical effect of electroacupuncture on depression is widely recognized. However, the signal transduction pathways and target proteins involved remain unclear. In the present study, rat models of chronic restraint stress were used to explore the mechanism by which electroacupuncture alleviates depression. Rats were randomly divided into control, model, and electroacupuncture groups. Chronic restraint stress was induced in the model and electroacupuncture groups by restraining rats for 28 days. In the electroacupuncture group, electroacupuncture pretreatment at Baihui（GV20） and Yintang（GV29） acupoints was performed daily（1 m A, 2 Hz, discontinuous wave, 20 minutes） prior to restraint for 28 days. Open field tests and body weight measurements were carried out to evaluate the depressive symptoms at specific time points. On day 28, the crossing number, rearing number, and body weights of the model group were significantly lower than those in the control group. Behavior test results indicated that rat models of depressive-like symptoms were successfully established by chronic restraint stress combined with solitary raising. On day 28, an isobaric tag for a relative and absolute quantitation-based quantitative proteomic approach was performed to identify differentially expressed proteins in hippocampal samples obtained from the model and electroacupuncture groups. The potential function of these differential proteins was predicted through the use of the Cluster of Orthologous Groups of proteins（COG） database. Twenty-seven differential proteins（uncharacteristic proteins expected） were selected from the model and electroacupuncture groups. In addition to unknown protein functions, COG are mainly concentrated in general prediction function, mechanism of signal transduction, amino acid transport and metabolism groups. This suggests that electroacupuncture improved depressive-like symptoms by regulating differential proteins, and most of these related proteins exist in nerve cells.

Differential Proteomics Analysis on Fluoride-resistant Streptococcus mutans by Label-free Quantitation

To figure out the changed biological characteristics of a fluoride-resistant strain compared with those of the parent sensitive strain of Streptococcus mutans（S,mutans） with the help of differential proteomics analysis,sixtyseven proteins with obvious differential expression were identified in fluoride-resistant strain.Among them are the important virulence factors causing caries,including glucosyltransferase-S,glucosyltransferase-I,glucosyltransferase-Si,major cell-surface adhesin Pac,F-ATPase β-subunit,Fth,molecular chaperone DnaK,chaperonin GroEL,and the lactate dehydrogenase.We obtained the differential proteomics expression profiles of the fluoride-resistant strain（UA159-FR） and its parental strain（UA159） of S.mutans.Several important virulence factors bringing about caries were identified to have differential expressions,among which the expression of lactate dehydrogenase in fluoride-resistant strain was enhanced compared to that in its parent strain.The results could provide the basis for analvsing the functions of proteins encoded by caries-related genes from the fluoride-resistant S.mutans strain.