ITRAQ-based comparative proteomic analysis of differences in the protein profiles of stems and leaves from two alfalfa genotypes
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ABSTRACT:
SUBMITTER: Sun H
PROVIDER: S-EPMC7525974 | biostudies-literature | 2020 Jan
REPOSITORIES: biostudies-literature
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Project description:To explore the molecular regulatory mechanisms of early stem and leaf development, proteomic analysis was performed on leaves and stems of F genotype alfalfa, with thin stems and small leaves, and M genotype alfalfa, with thick stems and large leaves.
2020-11-13 | PXD022535 | iProX
Project description:Cotton yields are greatly reduced under high salinity stress conditions, although cotton is considered a moderately salt-tolerant crop. Understanding at the molecular level how cotton responds to salt stress will help in developing salt tolerant varieties. Here, we combined physiological analysis with isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics of seedling leaves of 2 genotypes differing in salinity tolerance to 200 mM (18.3 dS/m) NaCl stress. Salt stress produced significant stress symptoms in the sensitive genotype Nan Dan Ba Di Da Hua (N), including lower relative water and chlorophyll contents and higher relative electrolyte leakage and Na+/K+ ratio in leaf samples, compared with those in the tolerant genotype Earlistaple 7 (Z). A total of 58 differentially abundant salt-responsive proteins were identified. Asp-Glu-Ala-Asp (DEAD)-box ATP-dependent RNA helicase 3 and protochlorophyllide reductase were markedly suppressed after salt treatment, whereas the phosphate-related differentially abundant proteins (DAPs) phosphoethanolamine N-methyltransferase 1 and 14-3-3-like protein E were induced, and all these proteins may play significant roles in salt stress. Twenty-nine salt-responsive proteins were also genotype specific, and 62.1 and 27.6% of these were related to chloroplast and defense responses, respectively. Based on the Arabidopsis thaliana protein interaction database, orthologs of 25 proteins showed interactions in Arabidopsis, and among these, a calmodulin protein was predicted to have 212 functional partners. In addition, the Golgi apparatus and calcium may be important for salt secretion in cotton. Through integrative proteome and transcriptome analysis, 16 DAPs were matched to differentially expressed genes and verified using qRT-PCR. On the basis of these findings, we proposed that some proteins related to chloroplast, ATP, ribosomal, and phosphate metabolism as well as to the Golgi apparatus and calcium may play key roles in the short-term salt stress response of cotton seedling leaves.
| S-EPMC5733471 | biostudies-literature
Project description:Cell walls are important for the growth and development of all plants. They are also valuable resources for feed and fiber, and more recently as a potential feedstock for bioenergy production. Cell wall proteins comprise only a fraction of the cell wall, but play important roles in establishing the walls and in the chemical interactions (e.g., crosslinking) of cell wall components. This crosslinking provides structure, but restricts digestibility of cell wall complex carbohydrates, limiting available energy in animal and bioenergy production systems. Manipulation of cell wall proteins could be a strategy to improve digestibility. An analysis of the cell wall proteome of apical alfalfa stems (less mature, more digestible) and basal alfalfa stems (more mature, less digestible) was conducted using a recently developed low-salt/density gradient method for the isolation of cell walls. Walls were subsequently subjected to a modified extraction utilizing EGTA to remove pectins, followed by a LiCl extraction to isolate more tightly bound proteins. Recovered proteins were identified using shotgun proteomics. We identified 272 proteins in the alfalfa stem cell wall proteome, 153 of which had not previously been identified in cell wall proteomic analyses. Nearly 70% of the identified proteins were predicted to be secreted, as would be expected for most cell wall proteins, an improvement over previously published studies using traditional cell wall isolation methods. A comparison of our and several other cell wall proteomic studies indicates little overlap in identified proteins among them, which may be largely due to differences in the tissues used as well as differences in experimental approach.
| S-EPMC3521126 | biostudies-literature
Project description:To reveal grain physio-chemical and proteomic differences between two barley genotypes, Zhenong8 and W6nk2 of high- and low-grain-Cd-accumulation, grain profiles of ultrastructure, amino acid and proteins were compared. Results showed that W6nk2 possesses significantly lower protein content, with hordein depicting the greatest genotypic difference, compared with Zhenong8, and lower amino acid contents with especially lower proportion of Glu, Tyr, Phe and Pro. Both scanning and transmission electron microscopy observation declared that the size of A-type starch molecule in W6nk2 was considerably larger than that of Zhenong8. Grains of Zhenong8 exhibited more protein-rich deposits around starch granules, with some A-type granules having surface pits. Seventeen proteins were identified in grains, using 2-DE coupled with mass spectrometry, with higher expression in Zhenong8 than that in W6nk2; including z-type serpin, serpin-Z7 and alpha-amylase/trypsin inhibitor CM, carbohydrate metabolism, protein synthesis and signal transduction related proteins. Twelve proteins were less expressed in Zhenong8 than that in W6nk2; including barley trypsin inhibitor chloroform/methanol-soluble protein (BTI-CMe2.1, BTI-CMe2.2), trypsin inhibitor, dehydroascorbate reductase (DHAR), pericentrin, dynein heavy chain and some antiviral related proteins. The data extend our understanding of mechanisms underlying Cd accumulation/tolerance and provides possible utilization of elite genetic resources in developing low-grain-Cd barley cultivars.
| S-EPMC3832469 | biostudies-literature
Project description:BACKGROUND: Alfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling. RESULTS: Cell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. In addition, 341,984 ESTs were generated from ES and PES internodes of genotype 773 using the GS FLX Titanium platform. The first alfalfa (Medicago sativa) gene index (MSGI 1.0) was assembled using the Sanger ESTs available from GenBank, the GS FLX Titanium EST sequences, and the de novo assembled Illumina sequences. MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1,294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Out of 55 SNPs randomly selected for experimental validation, 47 (85%) were polymorphic between the two genotypes. We also identified numerous allelic variations within each genotype. Digital gene expression analysis identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes. CONCLUSIONS: Our results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a forage crop and cellulosic feedstock.
| S-EPMC3112146 | biostudies-literature
Project description:Alfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. The application of genomic approaches would advance development of alfalfa as a cellulosic feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling. Cell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. These ESTs were de novo assembled into 132,153 unique sequences. By combining the de novo assembled ESTs (132,153 sequences) with our previously identified EST sequences (341,984 sequences, unpublished data), and the ESTs available from GenBank (12,371 sequences), we built the first Alfalfa Gene Index (MSGI 1.0). MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1, 294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Transcript profiling of stem internodes of genotypes 708 and 773 was conducted by quantifying the number of Illumina EST reads that were mapped to sequences in MSGI 1.0. We identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes. Our results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index (MSGI 1.0) assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a cellulosic feedstock.
2011-04-21 | GSE26757 | GEO
Project description:To investigate the protein profiling of buffalo oocytes at the germinal vesicle (GV) stage and metaphase II (MII) stage, an iTRAQ-based strategy was applied. A total of 3,763 proteins were identified, which representing the largest buffalo oocytes proteome dataset to date. Among these proteins identified, 173 proteins were differentially expressed in GV oocytes and competent MII oocytes, and 146 proteins were differentially abundant in competent and incompetent matured oocytes. Functional and KEGG pathway analysis revealed that the up-regulated proteins in competent MII oocytes were related to chromosome segregation, microtubule-based process, protein transport, oxidation reduction, ribosome, and oxidative phosphorylation, etc., in comparison with GV and incompetent MII oocytes. This is the first proteomic report on buffalo oocytes from different maturation stages and developmental competent status. These data will provide valuable information for understanding the molecular mechanism underlying buffalo oocyte maturation, and these proteins may potentially act as markers to predict developmental competence of buffalo oocyte during in vitro maturation.
| S-EPMC4999887 | biostudies-other
Project description:BACKGROUND: The GeneChip(R) Medicago Genome Array, developed for Medicago truncatula, is a suitable platform for transcript profiling in tetraploid alfalfa [Medicago sativa (L.) subsp. sativa]. However, previous research involving cross-species hybridization (CSH) has shown that sequence variation between two species can bias transcript profiling by decreasing sensitivity (number of expressed genes detected) and the accuracy of measuring fold-differences in gene expression. RESULTS: Transcript profiling using the Medicago GeneChip(R) was conducted with elongating stem (ES) and post-elongation stem (PES) internodes from alfalfa genotypes 252 and 1283 that differ in stem cell wall concentrations of cellulose and lignin. A protocol was developed that masked probes targeting inter-species variable (ISV) regions of alfalfa transcripts. A probe signal intensity threshold was selected that optimized both sensitivity and accuracy. After masking for both ISV regions and previously identified single-feature polymorphisms (SFPs), the number of differentially expressed genes between the two genotypes in both ES and PES internodes was approximately 2-fold greater than the number detected prior to masking. Regulatory genes, including transcription factor and receptor kinase genes that may play a role in development of secondary xylem, were significantly over-represented among genes up-regulated in 252 PES internodes compared to 1283 PES internodes. Several cell wall-related genes were also up-regulated in genotype 252 PES internodes. Real-time quantitative RT-PCR of differentially expressed regulatory and cell wall-related genes demonstrated increased sensitivity and accuracy after masking for both ISV regions and SFPs. Over 1,000 genes that were differentially expressed in ES and PES internodes of genotypes 252 and 1283 were mapped onto putative orthologous loci on M. truncatula chromosomes. Clustering simulation analysis of the differentially expressed genes suggested co-expression of some neighbouring genes on Medicago chromosomes. CONCLUSIONS: The problems associated with transcript profiling in alfalfa stems using the Medicago GeneChip as a CSH platform were mitigated by masking probes targeting ISV regions and SFPs. Using this masking protocol resulted in the identification of numerous candidate genes that may contribute to differences in cell wall concentration and composition of stems of two alfalfa genotypes.
| S-EPMC2893600 | biostudies-literature
Project description:Salinity severely threatens land use capability and crop yields worldwide. Understanding the mechanisms that protect soybeans from salt stress will help in the development of salt-stress tolerant leguminous plants. Here we initially analyzed the changes in malondialdehyde levels, the activities of superoxide dismutase and peroxidases, chlorophyll content, and Na(+)/K(+) ratios in leaves and roots from soybean seedlings treated with 200 mM NaCl at different time points. We found that the 200 mM NaCl treated for 12 h was optimal for undertaking a proteomic analysis on soybean seedlings. An iTRAQ-based proteomic approach was used to investigate the proteomes of soybean leaves and roots under salt treatment. These data are available via ProteomeXchange with the identifier PXD002851. In total, 278 and 440 proteins with significantly altered abundances were identified in leaves and roots of soybean, respectively. From these data, a total of 50 proteins were identified in the both tissues. These differentially expressed proteins (DEPs) were from 13 biological processes. Moreover, protein-protein interaction analysis revealed that proteins involved in metabolism, carbohydrate and energy metabolism, protein synthesis and redox homeostasis could be assigned to four high salt stress response networks. Furthermore, semi-quantitative RT-PCR analysis revealed that some of the proteins, such as a 14-3-3, MMK2, PP1, TRX-h, were also regulated by salt stress at the level of transcription. These results indicated that effective regulatory protein expression related to signaling, membrane and transport, stress defense and metabolism all played important roles in the short-term salt response of soybean seedlings.
| S-EPMC4850148 | biostudies-literature
Project description:The plant leaf, the main organ of photosynthesis, is an important regulator of growth. To explore the difference between leaf size of Populusdeltoides 'Danhong' (Pd) and Populus simonii 'Tongliao1' (Ps), we investigated the leaf length, leaf width, leaf thickness, leaf area, leaf mass per area (LMA), and cell size of leaves from two genotypes and profiled the transcriptome-wide gene expression patterns through RNA sequencing. Our results show that the leaf area of Pd was significantly larger than that of Ps, but the epidermal cell area was significantly smaller than that of Ps. The difference of leaf size was caused by cell numbers. Transcriptome analysis also revealed that genes related to chromosome replication and DNA repair were highly expressed in Pd, while genes such as the EXPANSIN (EXPA) family which promoted cell expansion were highly expressed in Ps. Further, we revealed that the growth-regulating factors (GRFs) played a key role in the difference of leaf size between two genotypes through regulation of cell proliferation. These data provide a valuable resource for understanding the leaf development of the Populus genus.
| S-EPMC8624656 | biostudies-literature