In-depth investigation of the soybean seed-filling proteome and comparison with a parallel study of rapeseed

To better understand the metabolic processes of seed filling in soybean (Glycine max), two complementary proteomic approaches, two-dimensional gel electrophoresis (2-DGE) and semicontinuous multidimensional protein identification technology (Sec-MudPIT) coupled with liquid chromatography-mass spectrometry, were employed to analyze whole seed proteins at five developmental stages. 2-DGE and Sec-MudPIT analyses collectively identified 478 nonredundant proteins with only 70 proteins common to both datasets. 2-DGE data revealed that 38% of identified proteins were represented by multiple 2-DGE species. Identified proteins belonged to 13 (2-DGE) and 15 (Sec-MudPIT) functional classes. Proteins involved in metabolism, protein destination and storage, and energy were highly represented, collectively accounting for 61.1% (2-DGE) and 42.2% (Sec-MudPIT) of total identified proteins. Membrane proteins, based upon transmembrane predictions, were 3-fold more prominent in Sec-MudPIT than 2-DGE. Data were integrated into an existing soybean proteome database (www.oilseedproteomics.missouri.edu). The integrated quantitative soybean database was compared to a parallel study of rapeseed (Brassica napus) to further understand the regulation of intermediary metabolism in protein-rich versus oil-rich seeds. Comparative analyses revealed (1) up to 3-fold higher expression of fatty acid biosynthetic proteins during seed filling in rapeseed compared to soybean; and (2) approximately a 48% higher number of protein species and a net 80% higher protein abundance for carbon assimilatory and glycolytic pathways leading to fatty acid synthesis in rapeseed versus soybean. Increased expression of glycolytic and fatty acid biosynthetic proteins in rapeseed compared to soybean suggests that a possible mechanistic basis for higher oil in rapeseed involves the concerted commitment of hexoses to glycolysis and eventual de novo fatty acid synthesis pathways.     

Dissecting the proteome of pea mature seeds reveals the phenotypic plasticity of seed protein composition

Pea (Pisum sativum L.) is the most cultivated European pulse crop and the pea seeds mainly serve as a protein source for monogastric animals. Because the seed protein composition impacts on seed nutritional value, we aimed at identifying the determinants of its variability. This paper presents the first pea mature seed proteome reference map, which includes 156 identified proteins (http://www.inra.fr/legumbase/peaseedmap/). This map provides a fine dissection of the pea seed storage protein composition revealing a large diversity of storage proteins resulting both from gene diversity and post‐translational processing. It gives new insights into the pea storage protein processing (especially 7S globulins) as a possible adaptation towards progressive mobilization of the proteins during germination. The nonstorage seed proteome revealed the presence of proteins involved in seed defense together with proteins preparing germination. The plasticity of the seed proteome was revealed for seeds produced in three successive years of cultivation, and 30% of the spots were affected by environmental variations. This work pinpoints seed proteins most affected by environment, highlighting new targets to stabilize storage protein composition that should be further analyzed.     

Assembly of the Trypanosoma brucei 60S ribosomal subunit nuclear export complex requires trypanosome-specific proteins P34 and P37

We previously identified two Trypanosoma brucei RNA binding proteins, P34 and P37, and determined that they are essential for proper ribosomal assembly in this organism. Loss of these proteins via RNA interference is lethal and causes a decrease in both 5S rRNA levels and formation of 80S ribosomes, concomitant with a decrease in total cellular protein synthesis. These data suggest that these proteins are involved at some point in the ribosomal biogenesis pathway. In the current study, we have performed subcellular fractionation in conjunction with immune capture experiments specific for 60S ribosomal proteins and accessory factors in order to determine when and where P34 and P37 are involved in the ribosomal biogenesis pathway. These studies demonstrate that P34 and P37 associate with the 60S ribosomal subunit at the stage of the nucleolar 90S particle and remain associated subsequent to nuclear export. In addition, P34 and P37 associate with conserved 60S ribosomal subunit nuclear export factors exportin 1 and Nmd3, suggesting that they are components of the 60S ribosomal subunit nuclear export complex in T. brucei. Most significantly, the pre-60S complex does not associate with exportin 1 or Nmd3 in the absence of P34 and P37. These results demonstrate that, although T. brucei 60S ribosomal subunits utilize a nuclear export complex similar to that described for other organisms, trypanosome-specific factors are essential to the process.     

Differential expressions of nuclear proteomes between honeybee (Apis mellifera L.) Queen and Worker Larvae: a deep insight into caste pathway decisions

Honeybees (Apis mellifera L.) possess individuals (castes) in their colonies, to which specific tasks are allocated. Owing to a difference in nutrition, the young female larvae develop into either a fertile queen or a sterile worker. Despite a series of investigations on the underlying mechanisms of honeybee caste polyphenism, information on proteins and enzymes involved in DNA and RNA regulation in the nucleus is still missing. The techniques of nuclear protein enrichment, two-dimensional electrophoresis, mass spectrometry and bioinformatics were applied to understand the nuclear proteome changes in response to changes in environmental settings (nutrition and time) during the early developmental stages at the third (72 h), fourth (96 h), and fifth (120 h) instars of the two caste intended larvae. A total of 120 differentially expressed nuclear proteins were identified in both caste intended larvae during these developmental stages. The third, fourth and fifth instars of queen prospective larvae expressed 69%, 84%, and 68% of the proteins that had altered expression, respectively. Particularly, the prospective queen larvae up-regulated most of the proteins with nuclear functions. In general, this changing nuclear proteome of the two caste intended larvae over the three developmental stages suggests variations in DNA and RNA regulating proteins and enzymes. These variations of proteins and enzymes involved in DNA and RNA regulation in response to differential nutrition between the two caste intended larvae lead the two caste larvae to pursue different developmental trajectories. Hence, this first data set of the nuclear proteome helps us to explore the innermost biological makings of queen and worker bee castes as early as before the 72 h (3rd instar). Also, it provides new insights into the honeybee's polymorphism at nuclear proteome level and paves new ways to understand mechanisms of caste decision in other eusocial insects.     

Large-scale analysis of the human ubiquitin-related proteome

Protein ubiquitylation contributes to the regulation of many cellular processes including protein degradation, receptor internalization, and repair of DNA damage. We now present a comprehensive characterization of ubiquitin-conjugated and ubiquitin-associated proteins in human cells. The proteins were purified by immunoaffinity chromatography under denaturing or native conditions. They were then digested with trypsin, and the resulting peptides were analyzed by 2-D LC and MS/MS. A total of 670 distinct proteins were identified; 345 proteins (51%) were classified as Urp-D (ubiquitin-related proteome under the denaturing condition) and comprised ubiquitin-conjugated molecules, whereas 325 proteins (49%) were classified as Urp-N (ubiquitin-related proteome only under the native condition) and included molecules that associated with ubiquitylated proteins. The proportions of proteins in various functional categories differed substantially between Urp-D and Urp-N. Many ribosomal subunits were detected in the Urp-D group of proteins and several of these subunits were directly shown to be ubiquitylated by mass spectrometric analysis, suggesting that ubiquitylation might play an important role in the regulation and/or quality control of ribosomal proteins. Our results demonstrate the potential of proteomics analysis of protein ubiquitylation to provide important insight into the regulation of protein stability and other ubiquitin-related cellular functions.     

The relationship between the spoT gene, the synthesis of stable RNA, ribosomal proteins, and the beta beta' subunits of RNA polymerase following a nutritional shiftup of Escherichia coli.

The level of ppGpp and rates of synthesis of stable RNA, ribosomal protein, and the beta and beta' subunits of RNA polymerase were measured following a nutritional shiftup in Escherichia coli strains, NF 929 (spoT+) and NF 930 (spoT-). In the spoT+ strain, ppGpp levels decreased 50% within 2 min following shiftup, and the rates of synthesis of stable RNA, ribosomal proteins, and the beta and beta' subunits of RNA polymerase increased with little or no lag. In contrast, in the spoT- strain, ppGpp levels transiently increased 40% during the first 6 min following shiftup. An inhibition in the rate of stable RNA synthesis and a delay in the increased synthesis of ribosomal proteins and beta and beta' subunits occurred concurrently with the transient increase in ppGpp. In addition, the DNA-dependent synthesis in vitro of the beta and beta' subunits of RNA polymerase was inhibited by physiological levels of ppGpp. Because of the timing and magnitude of the changes in ppGpp levels in the spoT- strain versus the timing when the new rates of stable RNA, ribosomal protein, and beta and beta' subunits synthesis are reached, it is concluded that ppGpp is not the sole element regulating the expression of these genes.     

Structure and function of the nucleolus.

The activity of the ribosomal RNA genes generates a distinct subnuclear structure, the nucleolus, which is the site of ribosome biogenesis. The signals that target proteins and snoRNAs (small nucleolar RNAs) to the nucleolus, the nuclear import of ribosomal proteins, the export of the completed ribosomal subunits and the molecular organization of the nucleolus have been the subject of intense research during the past year. Evidence is accumulating that nucleoli functionally interact with coiled bodies and are also involved in the maturation of non-ribosomal RNA species.     

Multiomics approach reveals a role of translational machinery in shaping maize kernel amino acid composition

Maize (Zea mays) seeds are a good source of protein, despite being deficient in several essential amino acids. However, eliminating the highly abundant but poorly balanced seed storage proteins has revealed that the regulation of seed amino acids is complex and does not rely on only a handful of proteins. In this study, we used two complementary omics-based approaches to shed light on the genes and biological processes that underlie the regulation of seed amino acid composition. We first conducted a genome-wide association study to identify candidate genes involved in the natural variation of seed protein-bound amino acids. We then used weighted gene correlation network analysis to associate protein expression with seed amino acid composition dynamics during kernel development and maturation. We found that almost half of the proteome was significantly reduced during kernel development and maturation, including several translational machinery components such as ribosomal proteins, which strongly suggests translational reprogramming. The reduction was significantly associated with a decrease in several amino acids, including lysine and methionine, pointing to their role in shaping the seed amino acid composition. When we compared the candidate gene lists generated from both approaches, we found a nonrandom overlap of 80 genes. A functional analysis of these genes showed a tight interconnected cluster dominated by translational machinery genes, especially ribosomal proteins, further supporting the role of translation dynamics in shaping seed amino acid composition. These findings strongly suggest that seed biofortification strategies that target the translation machinery dynamics should be considered and explored further.

An integrated approach reveals the key role of translational machinery in maize kernel amino acid natural variation and homeostasis, highlighting targets for seed amino acid biofortification.     

种子萌发的抑制调控机制

种子萌发是植物生命周期中一个重要的生理过程,激素作用、miRNA抑制、mRNA区域化、表观遗传调控等多个层次的分子抑制参与该过程的调控。赤霉素（解除抑制的激素）合成和失活的调控主要发生在转录水平,而脱落酸（引起抑制的激素）信号转导途径的调控则通过蛋白质抑制物的降解来实现。miRNA在转录后水平使其靶基因的mRNA降解,抑制种子的萌发;通过mRNA的区域化抑制与萌发相关基因的翻译属于另一层次的转录后抑制;小RNA介导的表观遗传机制也可能在种子萌发过程基因表达的协同调控中发挥重要作用。与分子水平的抑制类似,胚乳和种皮产生的机械抑制也很重要。     

Redox-sensitive proteome and antioxidant strategies in wheat seed dormancy control

Oxidative signalling by ROS has been demonstrated to play a role in seed dormancy alleviation, but the detailed molecular mechanisms underlying this process remain largely unknown. Here, we show dynamic differences in redox-sensitive proteome upon wheat seed dormancy release. Using thiol-specific fluorescent labelling, solubility-based protein fractionation, 2-D IEF PAGE, and MS analysis in conjunction with wheat EST sequence libraries, proteins with reversible oxidoreductive changes were characterized. Altogether, 193 reactive Cys were found in 79 unique proteins responding differentially in dormant, non-dormant, abscisic, or gibberellic acid-treated seed protein extracts from RL4137, a wheat cultivar with extreme dormancy. The identified proteins included groups that are redox-, stress-, and pathogen-responsive, involved in protein synthesis and storage, are enzymes of carbohydrate metabolism, proteases, and those involved in transport and signal transduction. Two types of redox response could be detected: (i) a dramatic increase in protein thiol redox state in seeds during imbibition and hormonal treatment; (ii) higher antioxidant capacity related to sensing of a threshold redox potential and balancing the existing redox pools, in dry dormant versus non-dormant seeds. These results highlight occurrence of the antioxidant defence mechanisms required for the protection of seed during a dormancy stage.