SMALL GRAIN 1, which encodes a mitogen‐activated protein kinase kinase 4, influences grain size in rice

Although grain size is one of the most important components of grain yield, little information is known about the mechanisms that determine final grain size in crops. Here we characterize rice small grain1 (smg1) mutants, which exhibit small and light grains, dense and erect panicles and comparatively slightly shorter plants. The short grain and panicle phenotypes of smg1 mutants are caused by a defect in cell proliferation. The smg1 mutations were identified, using a map‐based cloning approach, in mitogen‐activated protein kinase kinase 4 (OsMKK4). Relatively higher expression of OsMKK4/SMG1 was detected in younger organs than in older ones, consistent with its role in cell proliferation. Green fluorescent protein (GFP)–OsMKK4/SMG1 fusion proteins appear to be distributed ubiquitously in plant cells. Further results revealed that OsMKK4 influenced brassinosteroid (BR) responses and the expression of BR‐related genes. Thus, our findings have identified OsMKK4 as a factor for grain size, and suggest a possible link between the MAPK pathways and BRs in grain growth.     

OsMAPK6, a mitogen‐activated protein kinase,influences rice grain size and biomass production

Grain size is an important agronomic trait in determining grain yield. However, the molecular mechanisms that determine the final grain size are not well understood. Here, we report the functional analysis of a rice (Oryza sativa L.) mutant, dwarf and small grain1 (dsg1), which displays pleiotropic phenotypes, including small grains, dwarfism and erect leaves. Cytological observations revealed that the small grain and dwarfism of dsg1 were mainly caused by the inhibition of cell proliferation. Map‐based cloning revealed that DSG1 encoded a mitogen‐activated protein kinase (MAPK), OsMAPK6. OsMAPK6 was mainly located in the nucleus and cytoplasm, and was ubiquitously distributed in various organs, predominately in spikelets and spikelet hulls, consistent with its role in grain size and biomass production. As a functional kinase, OsMAPK6 interacts strongly with OsMKK4, indicating that OsMKK4 is likely to be the upstream MAPK kinase of OsMAPK6 in rice. In addition, hormone sensitivity tests indicated that the dsg1 mutant was less sensitive to brassinosteroids (BRs). The endogenous BR levels were reduced in dsg1, and the expression of several BR signaling pathway genes and feedback‐inhibited genes was altered in the dsg1 mutant, with or without exogenous BRs, indicating that OsMAPK6 may contribute to influence BR homeostasis and signaling. Thus, OsMAPK6, a MAPK, plays a pivotal role in grain size in rice, via cell proliferation, and BR signaling and homeostasis.     

The Regulator RamA Influences cmytA Transcription and Cell Morphology of Corynebacterium ammoniagenes

RamA plays a regulatory role for acetate utilization and S-layer biosynthesis in Corynebacterium glutamicum. Looking for any additional role, the function of RamA was analyzed in Corynebacterium ammoniagenes, which is closely related to C. glutamicum. In this study, we showed that the ΔramA mutant constructed by a markerless knockout strategy possessed increased cell surface hydrophobicity, leading to the formation of aggregated cell masses in liquid media. In addition, the mutant exhibited an elongated cell shape as observed by SEM, suggesting that cell wall-associated proteins might be influenced. Furthermore, cell surface proteome analysis revealed that the expression of cmytA gene encoding corynomycoloyl transferase required for cell wall biosynthesis was down-regulated in the mutant, supporting the regulatory role of RamA in cell wall assembly. These studies support a novel regulatory role of RamA in inducing the expression of proteins required for cell wall assembly.     

Quantitative proteomics identified 3 oxidative phosphorylation genes with clinical prognostic significance in gastric cancer

The aim of the present study was to explore the underlying mechanisms involved in gastric cancer (GC) formation using data‐independent acquisition (DIA) quantitative proteomics analysis. We identified the differences in protein expression and related functions involved in biological metabolic processes in GC. Totally, 745 differentially expressed proteins (DEPs) were found in GC tissues vs. gastric normal tissues. Despite enormous complexity in the details of the underlying regulatory network, we find that clusters of proteins from the DEPs were mainly involved in 38 pathways. All of the identified DEPs involved in oxidative phosphorylation were down‐regulated. Moreover, GC possesses significantly altered biological metabolic processes, such as NADH dehydrogenase complex assembly and tricarboxylic acid cycle, which is mostly consistent with that in KEGG analysis. Furthermore the higher expression of UQCRQ, NDUFB7 and UQCRC2 were positively correlated with a better prognosis, implicating these proteins may as novel candidate diagnostic and prognostic biomarkers.     

Proteomic understanding of intracellular responses of recombinant Chinese hamster ovary cells cultivated in serum-free medium supplemented with hydrolysates

In order to understand the intracellular responses in recombinant CHO (rCHO) cells producing antibody in serum-free medium (SFM) supplemented with optimized hydrolysates mixtures, yielding the highest specific growth rate (μ, SFM#S1) or the highest specific antibody productivity (q _Ab, SFM#S2), differentially expressed proteins in rCHO cells are measured by two-dimensional gel electrophoresis combined with nano-LC-ESI-Q-TOF tandem MS. The comparative proteomic analysis with basal SFM without hydrolysates revealed that the addition of hydrolysate mixtures significantly altered the profiles of CHO proteome. In SFM#S1, the expression of metabolism-related proteins, cytoskeleton-associated proteins, and proliferation-related proteins was up-regulated. On the other hand, the expression of anti-proliferative proteins and pro-apoptotic protein was down-regulated. In SFM#S2, the expression of various chaperone proteins and proliferation-linked proteins was altered. 2D-Western blot analysis of differentially expressed proteins confirmed the proteomic results. Taken together, identification of differentially expressed proteins in CHO cells by a proteomic approach can provide insights into understanding the effect of hydrolysates on intracellular events and clues to find candidate genes for cell engineering to maximize the protein production in rCHO cells.     

Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment

Strigolactones (SLs) are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica) to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development.     

Phenotypic and proteomic analysis of positively regulated gellan biosynthesis pathway in Sphingomonas elodea

Sphingomonas elodea is a Gram-negative bacterium capable of producing ‘gellan gum’ exopolysaccharide that is the most extensively studied expolysaccharides of microbial origin. In this study, we investigated the phenotypic and proteomic alterations in S. elodea by homogeneously expressing both gelA and gelN involved in positive regulation and extracellular secretion of metabolites in gellan biosynthesis, respectively. Expression of six histidine-tagged GelA and GelN was determined by Western blot analysis. Successful expression of GelA and GelN resulted in both morphological changes of colonies and enhanced secretion of gellan into the growth medium (GelA, 21.2% more and GelN, 48.3% more) overexpressed compared to the wile-type. Comparative two-dimensional gel electrophoresis analysis revealed a differential proteome expression in S. elodea overexpressing GelA and GelN. Proteins up- or down-regulated by GelA and GelN overexpression were found to be mainly sugar transportation proteins, two-component regulatory proteins, and proteins involved in secretion pathways. The results suggest that the effect of GelA and GelN overexpression on gellan biosynthesis might be mainly caused by increased transportation of sugar units or enhanced exportation of gellan.     

Novel insights into plasma biomarker candidates in patients with chronic mountain sickness based on proteomics

Chronic mountain sickness (CMS) is a progressive incapacitating syndrome induced by lifelong exposure to hypoxia. In the present study, proteomic analysis was used to identify the differentially expressed proteins (DEPs) and then evaluate the potential plasma biomarkers between CMS and non-CMS groups. A total of 145 DEPs were detected in CMS Han Chinese people who live in the plateau (CMS-HPu), among which 89 were significantly up-regulated and 56 were significantly down-regulated. GO enrichment analysis showed that various biological processes were enriched, including the hydrogen peroxide metabolic/catabolic process, reactive oxygen species (ROS) metabolic, and acute inflammatory response. Protein–protein interaction analysis showed that antioxidant activity, the hydrogen peroxide catabolic process and peroxidase activity were primarily mapped in interaction proteins. Nine modules showed significantly clustering based on WGCNA analysis, with two being the most significant, and GO analysis showed that proteins of both modules were primarily enriched in oxidative stress-related biological processes. Four DEPs increased in CMS patients were evaluated as the candidate biomarkers, and three showed significant AUC: hemoglobin β chain (HB-β), thioredoxin-1 (TRX1), and phosphoglycerate kinase 1 (PGK1). The present study provides insights into the pathogenesis of CMS and further evaluates the potentially biomarkers for its prevention and treatment of it.     

Proteome analysis of Desulfovibrio desulfuricans G20 mutants using the accurate mass and time (AMT) tag approach

Abundance values obtained from direct LC-MS analyses were used to compare the proteomes of six transposon-insertion mutants of Desulfovibrio desulfuricans G20, the lab strain (G20lab) and a sediment-adapted strain (G20sediment). Three mutations were in signal transduction histidine kinases, and three mutations were in other regulatory proteins. The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to analyze the proteomes. A total of 1318 proteins was identified with high confidence, approximately 35% of all predicted proteins in the D. desulfuricans G20 genome. Proteins from all functional categories were identified. Significant differences in the abundance of 30 proteins were detected between the G20lab strain and the G20sediment strain. Abundances of proteins for energy metabolism, ribosomal synthesis, membrane biosynthesis, transport, and flagellar synthesis were affected in the mutants. Specific examples of proteins down-regulated in mutants include a putative tungstate transport system substrate-binding protein and several proteins related to energy production, for example, 2-oxoacid:acceptor oxidoreductase, cytochrome c-553, and formate acetyltransferase. In addition, several signal transduction mechanism proteins were regulated in one mutant, and the abundances of ferritin and hybrid cluster protein were reduced in another mutant. However, the similar abundance of universal stress proteins, heat shock proteins, and chemotaxis proteins in the mutants revealed that regulation of chemotactic behavior and stress regulation might not be observed under our growth conditions. This study provides the first proteomic overview of several sediment fitness mutants of G20, and evidence for the difference between lab strains and sediment-adapted strains at the protein level.     

Identification and functional analysis of proteins in response to light intensity,temperature and water potential in Brassica rapa hypocotyl

Hypocotyl elongation is an early event in plant growth and development and is sensitive to fluctuations in light, temperature, water potential and nutrients. Most research on hypocotyl elongation has focused on the regulatory mechanism of a single environment factor. However, information about combined effects of multi‐environment factors remains unavailable, and overlapping sites of the environmental factors signaling pathways in the regulation of hypocotyl elongation remain unclear. To identify how cross‐talks among light intensity, temperature and water potential regulate hypocotyl elongation in Brassica rapa L. ssp. chinesis, a comprehensive isobaric tag for relative and absolute quantitation‐based proteomic approach was adopted. In total, 7259 proteins were quantified, and 378 differentially expressed proteins (DEPs) were responsive to all three environmental factors. The DEPs were involved in a variety of biochemical processes, including signal transduction, cytoskeletal organization, carbohydrate metabolism, cell wall organization, protein modification and transport. The DEPs did not function in isolation, but acted in a large and complex interaction network to affect hypocotyl elongation. Among the DEPs, phyB was outstanding for its significant fold change in quantity and complex interaction networks with other proteins. In addition, changes of sensitivity to environmental factors in phyB‐9 suggested a key role in the regulation of hypocotyl elongation. Overall, the data presented in this study show a profile of proteins interaction network in response to light intensity, temperature and water potential and provides molecular basis of hypocotyl elongation in B. rapa.     

Identification of Membrane-Associated Proteins Regulated by the Arbuscular Mycorrhizal Symbiosis

A sub-cellular proteomic approach was carried out to monitor membrane-associated protein modifications in response to the arbuscular mycorrhizal (AM) symbiosis. Membrane proteins were extracted from Medicago truncatula roots either inoculated or not with the AM fungus Glomus intraradices. Comparative two-dimensional electrophoresis revealed that 36 spots were differentially displayed in response to the fungal colonization including 15 proteins induced, 3 up-regulated and 18 down-regulated. Among them, seven proteins were found to be commonly down-regulated in AM-colonized and phosphate-fertilized roots. Twenty-five spots out of the 36 of interest could be identified by matrix assisted laser desorption/ionisation-time of flight and/or tandem mass spectrometry analyses. Excepting an acid phosphatase and a lectin, none of them was previously reported as being regulated during AM symbiosis. In addition, this proteomic approach allowed us for the first time to identify AM fungal proteins in planta.     

Intratympanic steroid treatment can reduce ROS and immune response in human perilymph investigated by in-depth proteome analysis

Intratympanic (IT) steroid treatment is one of the most widely used and effective treatments for inner ear disorders such as sudden sensorineural hearing loss (SNHL). However, a clear mechanism of IT steroids in inner ear recovery has not yet been revealed. Therefore, we investigated proteome changes in extracted human perilymph after steroid treatment. In this study, we applied a tandem mass spectrometry (MS/MS)-based proteomics approach to discover global proteome changes by comparing human perilymph after steroid treatment with non-treated perilymph group. Using liquid chromatography-MS/MS analysis, we selected 156 differentially expressed proteins (DEPs) that were statistically significant according to Student's t-test. Functional annotation analysis showed that upregulated proteins after steroid treatment are related to apoptosis signaling, as well as reactive oxygen species (ROS) and immune responses. The protein–protein interaction (PPI) clusters the proteins associated with these processes and attempts to observe signaling circuitry, which mediates cellular response after IT steroid treatments. Moreover, we also considered the interactome analysis of DEPs and observed that those with high interaction scores were categorized as having equivalent molecular functions (MFs). Collectively, we suggest that DEPs and interacting proteins in human perilymph after steroid treatment would inhibit the apoptotic and adaptive immune processes that may lead to anti-inflammatory effects.     

拟南芥RPP1A参与幼苗生长的蛋白质组学分析

核糖体蛋白不仅参与蛋白质合成,而且参与植物生长发育的调控.利用拟南芥核糖体磷酸蛋白P1(ribosomal phosphoprotein P1,RPP1)家族基因RPP1A缺失突变体rpp1a研究RPP1A缺失对幼苗蛋白质表达水平的影响,揭示其参与调控幼苗生长的作用机制.表型分析发现,与野生型WT相比,RPP1A缺失导...     

Protein dynamics during seed germination under copper stress in Arabidopsis over-expressing <Emphasis Type="Italic">Potentilla superoxide dismutase</Emphasis>

Copper (Cu), though an essential micronutrient for plants, poses toxicity at higher concentrations possibly by inducing oxidative stress. With the background that enzyme superoxide dismutase (SOD) ameliorates oxidative stress, the present work focused on understanding physiological and proteomic response of Arabidopsis seeds constitutively over-expressing copper–zinc SOD of Potentilla atrosanguinea (PaSOD) during germination in response to varied concentrations of copper sulphate (Cu stress). Transgenics showed higher germination percentage and required less “mean time to germination” under Cu-stress. In response to Cu stress, 39 differentially expressed protein spots were detected by 2-D electrophoresis in proteins of germinating wild type (WT) and transgenic seeds, of which 14 spots appeared exclusively in transgenics. Among the rest 25 protein spots, 14 showed down-regulation, one showed up-regulation, and 10 spots disappeared. MALDI-TOF and subsequent peptide mass fingerprinting analysis revealed that the down-regulated proteins in transgenics were related to oxidative stress, detoxification, germination, intermediary metabolism and regulatory proteins. Up-regulated proteins in WT and down-regulated proteins in transgenic during Cu stress were the same. Changes in key proteins, vis-à-vis alleviation of oxidative stress in transgenic Arabidopsis over-expressing PaSOD possibly alleviated toxicity of Cu-induced stress during seed germination, resulting in higher germination rate and germination percentage.