水稻胚胎发育时期的特异性蛋白质

对不同发育时期的水稻胚蛋白质,进行了单向SDS聚丙烯酰胺凝胶电泳和双向凝胶电泳分析。单向电泳图中存在20kD左右、胚分化完成期以后特有的蛋白质。根据双向电泳图谱,稻胚总蛋白按其在胚胎发育过程中的动态可分为7类,其中组成性的蛋白质约占85％,在不同发育时期基本不变;具有发育时期特异性的蛋白质约占15％。以分化期胚蛋白质的抗体,对各时期的胚蛋白质进行交叉免疫电泳,所得图谱总体上是相似的,但不同时期的图谱之间出现沉淀峰的峰形、位置和数目的差异。交叉免疫电泳图谱的这种变化可能反映了稻胚发育过程中某些分化期蛋白质发生的变化。     

Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn

The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.     

Proteomic profiles of embryonic development in the ascidian Ciona intestinalis

We report here proteomics-based protein profiles of three embryonic stages of the ascidian Ciona intestinalis. Two-dimensional gel electrophoresis revealed 416, 539, and 695 protein spots in the unfertilized eggs, 16 cell-stage embryos, and tadpole larvae, respectively. Comparative and quantitative analyses of the spot patterns identified proteins showing an increase or decrease in amount during embryonic development. Protein identification by MALDI-TOF/MS indicated not only the abundance and importance of metabolic enzymes and translation elongation factors but also the functional importance of actin-binding proteins and molecular chaperones during ascidian development. Global changes in spots for vitellogenin-like protein suggested post-translational modification or proteolytic digestion of this protein during embryogenesis. Comparison between mRNA and protein levels among unfertilized eggs, 16 cell-stage embryos and tadpole larvae indicated nonparallel expression patterns of genes and proteins. Ascidians provide an excellent system for studying gene expression and cell differentiation during development, and the present study should shed light on the associated molecular mechanism at the protein level.     

Proteomic characterization of the greening process in rice seedlings using the MS spectral intensity-based label free method

Illumination-induced greening in dark-grown plants is one of the most dramatic developmental processes known in plants. In our current study, we characterized the greening process of rice seedlings using comparative proteome analysis. We identified 886 different proteins in both whole cell lysates of illuminated and nonilluminated rice shoots and performed comparative proteome analysis based on the MS spectral intensities obtained for unique peptides from respective proteins. Furthermore, the changes in the levels of individual proteins were then compared with those of the corresponding mRNAs. The results revealed well-coordinated increases in the enzymes involved in the Calvin cycle at both the protein and mRNA levels during greening, and that the changes at the mRNA level precede those at the protein level. Although a much lower effect of illumination was found on the enzymes associated with glycolysis and the TCA cycle, coordinated increases during greening were evident for the enzymes involved in photorespiration and nitrogen assimilation as well as the components of the chloroplastic translational machinery. These results thus define the differential regulation of distinct biological systems during greening in rice and demonstrate the usefulness of comprehensive and comparative proteome analysis for the characterization of biological processes in plant cells.     

Comparative proteomics of tuber induction, development and maturation reveal the complexity of tuberization process in potato (Solanum tuberosum L.)

Tuberization in potato ( Solanum tuberosum L.) is a developmental process that serves a double function, as a storage organ and as a vegetative propagation system. It is a multistep, complex process and the underlying mechanisms governing these overlapping steps are not fully understood. To understand the molecular basis of tuberization in potato, a comparative proteomic approach has been applied to monitor differentially expressed proteins at different development stages using two-dimensional gel electrophoresis (2-DE). The differentially displayed proteomes revealed 219 protein spots that change their intensities more than 2.5-fold. The LC-ES-MS/MS analyses led to the identification of 97 differentially regulated proteins that include predicted and novel tuber-specific proteins. Nonhierarchical clustering revealed coexpression patterns of functionally similar proteins. The expression of reactive oxygen species catabolizing enzymes, viz., superoxide dismutase, ascorbate peroxidase and catalase, were induced by more than 2-fold indicating their possible role during the developmental transition from stolons into tubers. We demonstrate that nearly 100 proteins, some presumably associated with tuber cell differentiation, regulate diverse functions like protein biogenesis and storage, bioenergy and metabolism, and cell defense and rescue impinge on the complexity of tuber development in potato.     

Proteomic changes in rice leaves during development of field-grown rice plants

Of the numerous factors affecting rice yield, how solar radiation is transformed into biomass through rice leaves is the most important. We have analyzed proteomic changes in rice leaves collected from six different developing stages (vegetative to ripening). We studied protein expression profiles of rice leaves by running two-dimensional gel electrophoresis. Differential protein expression among the six phases were analyzed by image analysis, which allowed the identification of 49 significantly different gel spots. The spots were further verified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, in which 89.8% of them were confirmed to be rice proteins. Finally, we confirmed some of the interesting rice proteins by immunoblotting. Three major conclusions can be drawn from these experimental results. (i) Protein expression in rice leaves, at least for high or middle abundance proteins, is attenuated during growth (especially some chloroplast proteins). However, the change is slow and the expression profiles are relatively stable during rice development. (ii) Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a major protein in rice leaves, is expressed at constant levels at different growth stages. Interestingly, a high ratio of degradation of the RuBisCO large subunit was found in all samples. This was confirmed by two approaches, mass spectrometry and immunoblotting. The degraded fragments are similar to other digested products of RuBisCO mediated by free radials. (iii) The expression of antioxidant proteins such as superoxide dismutase and peroxidase decline at the early ripening stage.     

Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum

The pattern of proteins synthesized at different stages of differentiation of the slime mold Dictyostelium discoideum was studied by two-dimensional polyacrylamide gel electrophoresis. Of the approximately 400 proteins detected during growth and/or development, synthesis of most continued throughout differentiation. Approximately 100 proteins show changes in their relative rates of synthesis. During the transition from growth to interphase, the major change observed is reduction in the relative rate of synthesis of about 8 proteins. Few further changes are noticeable until the stage of late cell aggregation, when production of about 40 new proteins begins and synthesis of about 10 is reduced considerably. Thereafter, there are few changes in the pattern of protein synthesis. Major changes in the relative rates of synthesis of a number of proteins are found during culmination, but few culmination-specific proteins are observed. In an attempt to understand the molecular basis for these changes, mRNA was isolated from different stages of differentiation and translated in an improved wheat germ cell-free system; the products were resolved on two-dimensional gels. The ratio of total translatable mRNA to total cellular RNA is constant throughout growth and differentiation. Messenger RNAs for many, but not all, developmentally regulated proteins can be identified by translation in cell-free systems. Actin is the major protein synthesized by vegetative cells and by early differentiating cells. The threefold increase in the relative rate of synthesis of actin during the first 2 hr of differentiation and the decrease which occurs thereafter can be accounted for by parallel changes in the amount of translatable actin mRNA. Most of the changes in the pattern of protein synthesis which occur during the late aggregation and culmination stages can also be accounted for by parallel increases or decreases in the amounts of translatable mRNAs encoding these proteins. It is concluded that mRNAs do not appear in a translatable form before synthesis of the homologous protein begins, and that regulation of protein synthesis during development is primarily at the levels of production or destruction of mRNA.     

Proteomic analysis of shoot-borne root initiation in maize (Zea mays L.)

Postembryonically formed shoot-borne roots make up the major backbone of the adult maize root stock. In this study the abundant soluble proteins of the first node (coleoptilar node) of wild-type and mutant rtcs seedlings, which do not initiate crown roots, were compared at two early stages of crown root formation. In Coomassie Bluestained 2-D gels, representing soluble proteins of coleoptilar nodes 5 and 10 days after germination, 146 and 203 proteins were detected, respectively. Five differentially accumulated proteins (> two-fold change; t-test: 95% significance) were identified in 5-day-old and 14 differentially accumulated proteins in 10-day-old coleoptilar nodes of wild-type versus rtcs. All 19 differentially accumulated proteins were identified via ESI MS/MS mass spectrometry. Five differentially accumulated proteins, including a regulatory G-protein and a putative auxin-binding protein, were further analyzed at the RNA expression level. These experiments confirmed differential gene expression and revealed subtle developmental regulation of these genes during early coleoptilar node development. This study represents the first proteomic analysis of shoot-borne root initiation in cereals and will contribute to a better understanding of the molecular basis of this developmental process unique to cereals.     

Analysis of the protein expression profiling during rice callus differentiation under different plant hormone conditions

Plant hormones function to coordinate plant growth and development. While the plant hormones, mainly auxin and cytokinin, are exogenously added to various plant tissue cultures, their effects on the organogenesis are apparent, but little is known concerning the molecular mechanisms by which they function in cultured cells. Rice, as a model plant in monocots, is also suitable to tissue culture studies. Here, we used four types of regeneration mediums with different relative concentrations of cytokinin and auxin for rice callus differentiation, the calli at different differentiation stages were collected for proteomic analysis. 2-dimensional electrophoresis revealed that 213 protein spots significantly differentially expressed during callus differentiation under different hormone conditions. By using mass spectrometry, 183 differentially expressed protein spots were identified to match 157 unique proteins. Most of these differential proteins were cellular/metabolic process-related proteins, whose different expression patterns may be correlated with the cytokinin and auxin regulation. Several hormone-related proteins were prominently featured in differentiated calli as compared with the initiated calli, such as alpha-amylase isoforms, mannose-binding rice lectin, putative dehydration stress-induced protein, cysteine endopeptidase and cystatin. All these results provide a novel insight into how the two plant hormones effect the callus differentiation in rice on the proteomic level.     

Entamoeba histolytica: response of the parasite to metronidazole challenge on the levels of mRNA and protein expression

Entamoeba histolytica remains a cause of significant morbidity and mortality in many countries. Although metronidazole has been used for the treatment of amoebiasis for several decades, little is known on how the amoebae react to this challenge on the levels of mRNA and protein expression. In this study, we examined their response using a focused microarray, quantitative RT-PCR, and two-dimensional gel electrophoresis (2DE). The amoebae modestly increased the levels of mRNA coding for superoxide dismutase, peroxiredoxin, ferredoxin, thioredoxin reductase, and the galactose/N-acetylgalactosamine specific lectin light and heavy chains. The mRNAs encoding actin and the 70 kDa and 101 kDa heat shock proteins were decreased. All the changes occured within 1 h of exposition, with very little further changes. In addition, the proteome revealed only very few changes. Taken together, E. histolytica appears to make only modest mRNA and protein expression changes when confronted with an unknown chemical stress.     

Autoimmune regulator, Aire, is a novel regulator of chondrocyte differentiation

Chondrocyte differentiation is controlled by various regulators, such as Sox9 and Runx2, but the process is complex. To further understand the precise underlying molecular mechanisms of chondrocyte differentiation, we aimed to identify a novel regulatory factor of chondrocyte differentiation using gene expression profiles of micromass-cultured chondrocytes at different differentiation stages. From the results of microarray analysis, the autoimmune regulator, Aire, was identified as a novel regulator. Aire stable knockdown cells, and primary cultured chondrocytes obtained from Aire^−/− mice, showed reduced mRNA expression levels of chondrocyte-related genes. Over-expression of Aire induced the early stages of chondrocyte differentiation by facilitating expression of Bmp2. A ChIP assay revealed that Aire was recruited on an Airebinding site (T box) in the Bmp2 promoter region in the early stages of chondrocyte differentiation and histone methylation was modified. These results suggest that Aire can facilitate early chondrocyte differentiation by expression of Bmp2 through altering the histone modification status of the promoter region of Bmp2.     

Comprehensive transcriptome analysis of phytohormone biosynthesis and signaling genes in microspore/pollen and tapetum of rice

To investigate the involvement of phytohormones during rice microspore/pollen (MS/POL) development, endogenous levels of IAA, gibberellins (GAs), cytokinins (CKs) and abscisic acid (ABA) in the mature anther were analyzed. We also analyzed the global expression profiles of genes related to seven phytohormones, namely auxin, GAs, CKs, brassinosteroids, ethylene, ABA and jasmonic acids, in MS/POL and tapetum (TAP) using a 44K microarray combined with a laser microdissection technique (LM-array analysis). IAA and GA(4) accumulated in a much higher amount in the mature anther compared with the other tissues, while CKs and ABA did not. LM-array analysis revealed that sets of genes required for IAA and GA synthesis were coordinately expressed during the later stages of MS/POL development, suggesting that these genes are responsible for the massive accumulation of IAA and GA(4) in the mature anther. In contrast, genes for GA signaling were preferentially expressed during the early developmental stages of MS/POL and throughout TAP development, while their expression was down-regulated at the later stages of MS/POL development. In the case of auxin signaling genes, such mirror-imaged expression observed in GA synthesis and signaling genes was not observed. IAA receptor genes were mostly expressed during the late stages of MS/POL development, and various sets of AUX/IAA and ARF genes were expressed during the different stages of MS/POL or TAP development. Such cell type-specific expression profiles of phytohormone biosynthesis and signaling genes demonstrate the validity and importance of analyzing the expression of phytohormone-related genes in individual cell types independently of other cells/tissues.