Dataset Information

Gel-free proteomic analysis of soybean root proteins affected by calcium under flooding stress.

ABSTRACT: Soybean is sensitive to flooding stress and exhibits reduced growth under flooding conditions. To better understand the flooding-responsive mechanisms of soybean, the effect of exogenous calcium on flooding-stressed soybeans was analyzed using proteomic technique. An increase in exogenous calcium levels enhanced soybean root elongation and suppressed the cell death of root tip under flooding stress. Proteins were extracted from the roots of 4-day-old soybean seedlings exposed to flooding stress without or with calcium for 2 days and analyzed using gel-free proteomic technique. Proteins involved in protein degradation/synthesis/posttranslational modification, hormone/cell wall metabolisms, and DNA synthesis were decreased by flooding stress; however, their reductions were recovered by calcium treatment. Development, lipid metabolism, and signaling-related proteins were increased in soybean roots when calcium was supplied under flooding stress. Fermentation and glycolysis-related proteins were increased in response to flooding; however, these proteins were not affected by calcium supplementation. Furthermore, urease and copper chaperone proteins exhibited similar profiles in 4-day-old untreated soybeans and 4-day-old soybeans exposed to flooding for 2 days in the presence of calcium. These results suggest that calcium might affect the cell wall/hormone metabolisms, protein degradation/synthesis, and DNA synthesis in soybean roots under flooding stress.

SUBMITTER: Oh M

PROVIDER: S-EPMC4202786 | biostudies-other | 2014

REPOSITORIES: biostudies-other

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Gel-free proteomic analysis of soybean root proteins affected by calcium under flooding stress.

Oh MyeongWon M Nanjo Yohei Y Komatsu Setsuko S

Frontiers in plant science 20141020

Soybean is sensitive to flooding stress and exhibits reduced growth under flooding conditions. To better understand the flooding-responsive mechanisms of soybean, the effect of exogenous calcium on flooding-stressed soybeans was analyzed using proteomic technique. An increase in exogenous calcium levels enhanced soybean root elongation and suppressed the cell death of root tip under flooding stress. Proteins were extracted from the roots of 4-day-old soybean seedlings exposed to flooding stress ...[more]

PMID: 25368623

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Project description:Flooding is a serious problem for soybean cultivation because it markedly reduces growth. We found that treatment with ABA enhances survival ratio of soybean under flooding stress. To investigate the role of ABA in soybean under flooding stress, proteomic changes were analyzed. Total protein and nuclear protein fractions from root tips of soybean seedlings that treated with flooding or flooding with ABA were analyzed. Protein samples were cleaned up with chloroform/methanol method and then subjected to in-solution digestion with trypsin and lysyl endopeptidase. Resulting peptides were analyzed on a nanospray LTQ XL Orbitrap MS (Thermo Fisher Scientific) operated in data-dependent acquisition mode with the installed Xcalibur software (version 2.0.7, Thermo Fisher Scientific). Using an Ultimate 3,000 nanoLC system (Dionex), peptides in 0.1% formic acid were loaded onto a C18 PepMap trap column (300 µm ID × 5 mm, Dionex). The peptides were eluted from the trap column and their separation and spraying were done using 0.1% formic acid in acetonitrile at a flow rate of 200 nL/min on a C18 Tip column (75 µm 1D × 120 mm, nano HPLC capillary column, NTTC-360/75-3, Nikkyo Technos) with a spray voltage of 1.5 kV. The elution was done with a linear acetonitrile gradient 8-30% in 120 min for gel free proteomics in 0.1% formic acid. Full-scan mass spectra were acquired in the Orbitrap over a mass range of 400-1,500 m/z with a resolution of 30,000. A lock mass function was used to obtain high mass accuracy. The top 10 most intense precursor ions were selected for collision-induced fragmentation in the linear ion trap at normalized collision energy of 35%. Dynamic exclusion was employed within 90 sec to prevent repetitive selection of peptides.Identification of proteins were performed by Mascot search engine (version 2.3.0.2, Matrix Science) using soybean peptide database (55,787 sequences) obtained from the soybean genome database (Phytozome version 8.0)and a common contaminants database (262 sequences). Parameters used in Mascot searches were follows: Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine was set as a variable modification. Trypsin was specified as the proteolytic enzyme and one missed cleavage was allowed. Peptide mass tolerance was set at 5 ppm, fragment mass tolerance was set at 0.5 Da, and peptide charge was set at +2, +3, and +4. Automatic decoy database search was performed in the search. Mascot results were filtered with Mascot percolator to improve accuracy and sensitivity in the peptide identification. False discovery rates for peptide identification of all searches were less than 1.0%. The Mascot results were used for differential analysis using SIEVE software (version 2.0, Thermo Fisher Scientific).

Dataset Information

Gel-free proteomic analysis of soybean root proteins affected by calcium under flooding stress.

Publications

Gel-free proteomic analysis of soybean root proteins affected by calcium under flooding stress.

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