Differential proteomic analysis during the vegetative phase change and the floral transition in Malus domestica

In order to identify the proteomic changes of apple (Malus domestica Borkh.) during the vegetative phase change and the floral transition, leaf protein of juvenile, adult vegetative and reproductive phase in a seedling ('Jonathan' × 'Golden Delicious') was extracted and analyzed by 2-D electrophoresis and Matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Seventy two gel spots with significant expression differences between ontogenetic phases were obtained. Five protein spots were only detected in leaves of juvenile phase and 11 were not; 17 spots were found exclusively in adult vegetative leaves; and only one spot solely appeared in reproductive leaves while 12 did not. Twenty six of the differentially expressed proteins identified were involved in photosynthesis. Seven enzymes were related to respiration and carbohydrate metabolism. Fifteen other proteins also presented qualitative or quantitative differences among developmental phases. The spatial distribution of one differentially expressed protein, serine hydroxymethyltransferase, was confirmed by enzyme linked immunosorbent assay and immunohistochemistry. These results strongly support the idea that the vegetative phase change and the floral transition are regulated independently during developmental process.     

miRNA control of vegetative phase change in trees

After germination, plants enter juvenile vegetative phase and then transition to an adult vegetative phase before producing reproductive structures. The character and timing of the juvenile-to-adult transition vary widely between species. In annual plants, this transition occurs soon after germination and usually involves relatively minor morphological changes, whereas in trees and other perennial woody plants it occurs after months or years and can involve major changes in shoot architecture. Whether this transition is controlled by the same mechanism in annual and perennial plants is unknown. In the annual forb Arabidopsis thaliana and in maize (Zea mays), vegetative phase change is controlled by the sequential activity of microRNAs miR156 and miR172. miR156 is highly abundant in seedlings and decreases during the juvenile-to-adult transition, while miR172 has an opposite expression pattern. We observed similar changes in the expression of these genes in woody species with highly differentiated, well-characterized juvenile and adult phases (Acacia confusa, Acacia colei, Eucalyptus globulus, Hedera helix, Quercus acutissima), as well as in the tree Populus x canadensis, where vegetative phase change is marked by relatively minor changes in leaf morphology and internode length. Overexpression of miR156 in transgenic P. x canadensis reduced the expression of miR156-targeted SPL genes and miR172, and it drastically prolonged the juvenile phase. Our results indicate that miR156 is an evolutionarily conserved regulator of vegetative phase change in both annual herbaceous plants and perennial trees.     

Gibberellins promote vegetative phase change and reproductive maturity in maize.

Postembryonic shoot development in maize (Zea mays L.) is divided into a juvenile vegetative phase, an adult vegetative phase, and a reproductive phase that differ in the expression of many morphological traits. A reduction in the endogenous levels of bioactive gibberellins (GAs) conditioned by any one of the dwarf1, dwarf3, dwarf5, or another ear1 mutations in maize delays the transition from juvenile vegetative to adult vegetative development and from adult vegetative to reproductive development. Mutant plants cease producing juvenile traits (e.g. epicuticular wax) and begin producing adult traits (e.g. epidermal hairs) later than wild-type plants. They also cease producing leaves and begin producing reproductive structures later than wild-type plants. These mutations greatly enhance most aspects of the phenotype of Teopod1 and Teopod2, suggesting that GAs suppress part but not all of the Teopod phenotype. Application of GA3 to Teopod2 mutants and Teopod1, dwarf3 double mutants confirms this result. We conclude that GAs act in conjunction with several other factors to promote both vegetative and reproductive maturation but affect different developmental phases unequally. Furthermore, the GAs that regulate vegetative and reproductive maturation, like those responsible for stem elongation, are downstream of GA20 in the GA biosynthetic pathway.     

The role of miR156 in developmental transitions in Nicotiana tabacum

Plants undergo a series of developmental transitions during their life cycle. After seed germination, plants pass through two distinct phases: the vegetative phase in which leaves are produced and the reproductive phase in which flowering occurs. Based on the reproductive competence and morphological changes, the vegetative phase can be further divided into juvenile and adult phases. Here, we demonstrate that the difference between juvenile and adult phase of Nicotiana tabacum is characterized by the changes in leaf size, leaf shape as well as the number of leaf epidermal hairs(trichomes). We further show that miR156, an age-regulated microR NA, regulates juvenile-to-adult phase transition in N. tabacum. Overexpression of miR156 results in delayed juvenile-to-adult transition and flowering. Together, our results support an evolutionarily conserved role of miR156 in plant developmental transitions.     

The liguleless2 gene of maize functions during the transition from the vegetative to the reproductive shoot apex

During a maize plant's (Zea mays) development, the shoot apical meristem (SAM) generates an apex that proceeds through different phases: juvenile vegetative, adult vegetative and reproductive. During each phase the structures produced are distinguishable from structures produced during the other phases. In this paper, we demonstrate that the LIGULELESS2 (LG2) function is required for an accurate vegetative to reproductive phase transition. The maize gene liguleless2 (lg2) has been shown to encode a basic-leucine zipper (bZIP) protein and to function in narrowing the region from which the ligule and auricle develop in a typical maize leaf. Here we show that lg2 mutant plants can have reduced long tassel branches, extra vegetative leaves and extra husk leaves when compared to wild-type siblings. This indicates a role for the lg2 gene in the vegetative to reproductive phase transition of the shoot apex. We also discuss a potential role for the lg2 gene in general phase transition processes.     

Ontogenetic trends in the morphological features of main stem annual shoots of Pinus pinaster (Pinaceae)

Phase change refers to the transition between juvenile and adult vegetative phases. The study of trees throughout their entire life span requires retrospective analyses and validates the use of a chronosequence by sequencing observations at different and successive stages. The main axis growth pattern of 62 maritime pines (Pinus pinaster) selected in a chronosequence of three stands consisting of 8-, 22-, and 48-yr-old trees was analyzed retrospectively. Comparison of measured features (length, number of axillary products, reproductive organs) at common ages from the three stands supported the validity of using these data to form a continuous chronosequence. Endogenous trends in tree development are revealed free from variability due to annual growth conditions. Two main phases of development corresponding respectively to the juvenile vegetative and adult reproductive stages were identified, and the transition between both occurred in 9-yr-old trees. The relevance of these two phases and more generally the notion of phase changes are discussed in light of observed trends in the values of studied growth and branching parameters that may either show gradual variations (such as length of annual shoot) or a distinctive expression in one or the other phase (such as presence of female cones).     

Comparative proteomic analysis of hearts of adult SCNT Bama miniature pigs (Sus scrofa)

This study aims to determine the effects of SCNT on cardiac development of SCNT pigs through proteomic methods. Heart proteins from three adult SCNTs and two normal reproductive Bama miniature pigs were extracted, separated, and identified via comparative proteomic methods, including two-dimensional gel electrophoresis, mass spectrometry, and Western blot. Eleven differentially expressed spots were identified as differentially expressed proteins, of which five spots were upregulated proteins such as cardiac myosin heavy chain, cathepsin D, and heat shock protein beta-1 (HSP27). By contrast, six spots were downregulated proteins such as alpha skeletal muscle and actin. The results also demonstrated that nuclear transfer might result in abnormal expression of some important proteins in hearts from SCNT pigs, and affect the cardiac development in SCNT pigs' survival.     

Analysis of the budding yeast pH 4–7 proteome in meiosis

Meiosis, the developmental programme generating haploid gametes from diploid precursors, requires two cell divisions and many innovations. In budding yeast, a large number of genes are expressed exclusively during meiosis while others are repressed compared to vegetative growth. Microarray analysis has shown that gene expression during meiosis is highly regulated, and has been used to classify yeast genes according to meiotic temporal expression pattern. In this study, we have begun to investigate the kinetics of meiotic protein expression using a proteomics approach. 2‐D DIGE was used to characterise the temporal protein expression patterns of the budding yeast pH 4–7 proteome in meiosis. More than 1400 meiotic protein spots were visualised and at least 63 spots were temporally regulated during meiosis in a statistically significant manner. Gel spots with significant expression changes were excised and 26 unique proteins were identified using LC‐MS/MS. The identified proteins could be classified into functional categories and the genes encoding a number of these were previously shown to be involved in yeast sporulation and meiosis. This data set was used to assemble the first differential 2‐D PAGE map of budding yeast meiosis, which can be accessed through a web server. This work represents one of the first quantitative proteomic analyses of meiosis in yeast and will provide a valuable resource for future investigations.     

The Arabidopsis compact inflorescence genes: phase-specific growth regulation and the determination of inflorescence architecture

During their life cycle, higher plants pass through a series of growth phases that are characterized by the production of morphologically distinct vegetative and reproductive organs and by different growth patterns. Three major phases have been described in Arabidopsis: juvenile vegetative, adult vegetative, and reproductive. In this report we describe a novel, phase-specific mutant in Arabidopsis, compact inflorescence (cif). The most apparent aspect of the cif phenotype is a strong reduction in the elongation of internodes in the inflorescence, resulting in the formation of a floral cluster at the apical end of all reproductive shoots. Elongation and expansion of adult vegetative rosette leaves are also compromised in mutant plants. The onset of the cif trait correlates closely with morphological changes marking the phase transition from juvenile to adult, and mutant plants produce normal flowers and are fully fertile. Hence the cif phenotype appears to be adult vegetative phase-specific. Histological sections of mutant inflorescence internodes indicate normal tissue specification, but reduced cell elongation compared to wild-type. compact inflorescence is inherited as a two-gene trait involving the action of a recessive and a dominant locus. These two cif genes appear to be key components of a growth regulatory pathway that is closely linked to phase change, and specifies critical aspects of plant growth and architecture including inflorescence internode length.     

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.     

强休眠玉米种子休眠前后的蛋白差异表达

以强休眠玉米自交系08-641为试验材料,分别对处于休眠状态下的新鲜收获种子和经过10 d后熟作用破除休眠的种子进行了蛋白质组学差异表达分析。结果表明,通过双向电泳技术在3次重复试验下休眠状态的08-641鲜种子蛋白2-DE图谱上共检测到约600个蛋白质点,在经过10 d后熟作用破除休眠的08-641种子蛋白2-DE图谱上共检测到约620个蛋白质点,其中下调表达蛋白质点4个,上调表达蛋白质点4个,新增蛋白质点8个,缺失表达蛋白质点7个。经过质谱鉴定的差异表达蛋白质主要涉及球蛋白、胚胎晚期丰富蛋白、豆球蛋白等贮藏物蛋白质;蛋白酶体、山梨醇脱氢酶等参与物质代谢的蛋白质;热激蛋白等参与蛋白质结构、细胞功能调控的蛋白质。推测08-641种子休眠是由于种子内休眠相关蛋白的过量表达或缺失抑制了种子的正常萌发。     

Dynamic proteome changes of Shigella flexneri 2a during transition from exponential growth to stationary phase

Shigella flexneri is an infectious pathogen that causes dysentery to human, which remains a serious threat to public health, particularly in developing countries. In this study, the global protein expression patterns of S. flexneri during transition from exponential growth to stationary phase in vitro were analyzed by using 2-D PAGE combined with MALDI-TOF MS. In a time-course experiment with five time points, the relative abundance of 49 protein spots varied significantly. Interestingly, a putative outer membrane protein YciD （OmpW） was almost not detected in the exponential growth phase but became one of the most abundant proteins in the whole stationary-phase proteome. Some proteins regulated by the global regulator FNR were also significantly induced （such as AnsB, AspA, FrdAB, and KatG） or repressed （such as AceEF, OmpX, SodA, and SucAB） during the growth phase transition. These proteins may be the key effectors of the bacterial cell cycle or play important roles in the cellular maintenance and stress responses. Our expression profile data provide valuable information for the study of bacterial physiology and form the basis for future proteomic analyses of this pathogen.     

2-D DIGE as a quantitative tool for investigating the HUPO Brain Proteome Project mouse series

Brain development and aging is a complex process involving proliferation, differentiation and apoptosis. Elucidating proteome changes in these processes can help to understand the mechanisms of brain development and maintenance as well as neurodegenerative diseases. The research reported here is a contribution to the HUPO Brain Proteome Project mouse pilot study. Whole, frozen C57BL/6J mouse brain comprising three different developmental stages (embryonic day 16, postnatal day 7, and postnatal days 54-58) were processed by using 2-D DIGE. A total of 1999 spots were matched between all gels. Of these, 206 spots were differentially expressed between the different stages: 122 spots were highest in intensity in embryonic stage E16, 26 highest in the juvenile group P7 and 58 spots highest in P56, the adult stage. The results show a pattern of temporal expression. Based on the expression patterns we tentatively suggest that proteins involved in the establishment of primary structures in the brain are expressed highest in the embryonic mouse. Proteins involved in the development of the brain are expressed highest in the juvenile phase and proteins that make utilization of the brain possible by delivering energy are expressed highest in the adult mice.     

Proteomic analysis of human serum by two-dimensional differential gel electrophoresis after depletion of high-abundant proteins

Two-dimensional differential gel electrophoresis (2-D DIGE) was used to analyze human serum following the removal of albumin and five other high-abundant serum proteins. After protein removal, serum was analyzed by SDS-PAGE as a preliminary screen, and significant differences between four high-abundant protein removal methods were observed. Antibody-based albumin removal and high-abundant protein removal methods were found to be efficient and specific. To further characterize serum after protein removal, 2-D DIGE was employed, enabling multiplexed analysis of serum through the use of three fluorescent protein dyes. Comparison between crude serum and serum after removal of high-abundant proteins clearly illustrates an increase in the number of lower abundant protein spots observed. Approximately 850 protein spots were detected in crude serum whereas over 1500 protein spots were exposed following removal of six high-abundant proteins, representing a 76% increase in protein spot detection. Several proteins that showed a 2-fold increase in intensity after depletion of high-abundant proteins, as well as proteins that were depleted during abundant protein removal methods, were further characterized by mass spectrometry. This series of experiments demonstrates that high-abundant protein removal, combined with 2-D DIGE, is a practical approach for enriching and characterizing lower abundant proteins in human serum. Consequently, this methodology offers advances in proteomic characterization, and therefore, in the identification of biomarkers from human serum.     

青海高原春小麦PEG胁迫与复水后叶片差异蛋白质组学研究

春小麦是青海省的主要粮食作物,青海高原干旱频繁且严重,尤以春旱为首,对小麦生长发育造成严重影响．为了从蛋白质组学水平分析小麦对干旱胁迫的应答特征,探讨小麦可能的抗旱机制,本研究对青海主栽春小麦品种青春38幼苗进行聚乙二醇 (polyethylene glycol,PEG) 6000胁迫和复水处理,采用IEF/SDS PAGE双向凝胶电泳技术,对PEG胁迫和复水处理的小麦叶片总蛋白质分别与正常浇水的对照进行差异蛋白质组学研究,经考马斯亮蓝G 250染色获得清晰度和重复性较好的双向电泳图谱．PD Quest软件处理分析各对照和处理图谱,在等电点4.0～7.0线性范围内,均可识别650个以上清晰蛋白质点,获得比较明显的差异表达蛋白点43个,其中8个蛋白点重复.从35个差异蛋白点中选取24个差异点进行MALDI-TOF-TOF-MS肽质量指纹图谱分析,应用Mascot软件在NCBInr数据库中搜索鉴定蛋白质,得到22个阳性结果．对鉴定得到的差异表达蛋白进行功能分析,它们分别参与了光合作用、蛋白质合成、能量代谢途径、细胞防御、氧化还原、运输、信号转导等过程,而且根据差异表达蛋白功能分类所占比例,发现干旱胁迫与光合作用关系最为紧密.     

Potential polyphenol markers of phase change in apple (Malus domestica)

In order to identify potential biochemical markers that can be used as indicators for phase change, the dynamics of polyphenolic compounds across apple seedlings (Malus domestica, Jonathan x Golden Delicious) were analyzed in this study by high performance liquid chromatography. Precocious flowering was induced by foliar sprays of plant growth regulators. Qualitative changes in the concentration of polyphenols were observed at node nos. 50, 80 and 120. Spontaneous and induced flowering was found at node nos. 122 and 77. It was reasonable to conclude that node no. 77 represented the point of transition between the juvenile phase and the adult vegetative phase, which was marked by the presence of phloridzin in the buds. The disappearance of myricitrin in the bark and the absence of caffeic acid in the aboveground tissues were qualitative markers of the reproductive phase, which was reached at node no. 122.