Article pubs.acs.org/jpr
Quantitative Proteomics Reveals the Flooding-Tolerance Mechanism in Mutant and Abscisic Acid-Treated Soybean Xiaojian Yin,†,‡ Minoru Nishimura,§ Makita Hajika,‡ and Setsuko Komatsu*,†,‡ †
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan National Institute of Crop Science, National Agriculture and Food Research Organization, Tsukuba 305-8518, Japan § Graduate School of Life and Food Sciences, Niigata University, Niigata 950-2181, Japan ‡
S Supporting Information *
ABSTRACT: Flooding negatively affects the growth of soybean, and several floodingspecific stress responses have been identified; however, the mechanisms underlying flooding tolerance in soybean remain unclear. To explore the initial flooding tolerance mechanisms in soybean, flooding-tolerant mutant and abscisic acid (ABA)-treated plants were analyzed. In the mutant and ABA-treated soybeans, 146 proteins were commonly changed at the initial flooding stress. Among the identified proteins, protein synthesis-related proteins, including nascent polypeptide-associated complex and chaperonin 20, and RNA regulation-related proteins were increased in abundance both at protein and mRNA expression. However, these proteins identified at the initial flooding stress were not significantly changed during survival stages under continuous flooding. Cluster analysis indicated that glycolysis- and cell wall-related proteins, such as enolase and polygalacturonase inhibiting protein, were increased in abundance during survival stages. Furthermore, lignification of root tissue was improved even under flooding stress. Taken together, these results suggest that protein synthesis- and RNA regulation-related proteins play a key role in triggering tolerance to the initial flooding stress in soybean. Furthermore, the integrity of cell wall and balance of glycolysis might be important factors for promoting tolerance of soybean root to flooding stress during survival stages. KEYWORDS: soybean, mutant, abscisic acid, flooding stress, root, stress tolerance
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INTRODUCTION Flooding is a widespread environmental phenomenon that decreases the survival rate of most terrestrial plants1 and is a major abiotic stress against crops.2 Under flooding conditions, the oxygen diffusion rate into the soil is drastically reduced compared to that in air.3 Furthermore, flooding lowers the soil redox potential, which limits the availability of soil nutrients.4 Among crops, soybean is a major source of oil/protein and is cultivated worldwide;5 however, it exhibits marked reductions in plant growth and grain yields in response to flooding stress.6,7 For this reason, the development of flooding-tolerant cultivars of soybean is desirable. To promote the agricultural development of soybean, uncovering the mechanism underlying flooding tolerance is necessary. In response to several days of flooding exposure, seedling root elongation and lateral root development of soybean are suppressed.8 Komatsu et al.9 reported that a flooding-tolerant soybean mutant line was developed through gamma-ray irradiation and demonstrated that root development was not delayed in the mutant under flooding conditions. Notably, the mutant line survived under flooding stress for 6 days and grew well after removal of the excess water. Furthermore, a comparison of flooding-responsive proteins between the mutant and wild-type indicated that suppression of glycolysis © XXXX American Chemical Society
and programmed cell death was important for the acquisition of flooding tolerance.9 Komatsu et al.10 also reported that the survival ratio was improved by treatment with abscisic acid (ABA). Analysis of proteins from flooding-treated soybean with supplemental ABA indicated that ABA enhanced flooding tolerance by controlling energy conservation through the regulation of several transcription-related proteins.10 These two findings provide suitable materials such as mutant line and ABA-treated soybean to study the flooding-tolerant mechanism. Under flooding stress, soybean root elongation is suppressed within 24 h.11 Analysis of soybean gene expression indicated that genes associated with alcohol fermentation, ethylene biosynthesis, and pathogen defense were regulated in the first 12 h of flooding stress.12 In addition, genes related to glycolysis and ubiquitin-mediated protein degradation were up-regulated; however, cell wall synthesis- and chromatin structure synthesisrelated genes were down-regulated during the first 6 h of flooding stress.13 Furthermore, the fresh weight and ATP content of soybean significantly increase and decrease, respectively, after only 3 h of flooding stress,14 indicating that soybean rapidly responds to initial flooding stress. Proteomic Received: March 3, 2016
A
DOI: 10.1021/acs.jproteome.6b00196 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research
darkness, and digested with trypsin and lysyl endopeptidase (Wako, Osaka, Japan) at 1:100 enzyme/protein concentration at 37 °C for 16 h. The peptides were acidified with formic acid (pH < 3), and the mixed solution was centrifuged at 20 000g for 10 min. The supernatant was collected as digested peptides. The peptide concentration was measured using Direct Detect Spectrometer (Millipore, Billerica, MA, USA).
studies have also revealed that ABA plays an important role in mediating flooding stress responses in soybean root tip through regulation of protein phosphorylation.15 The results from these transcriptomic and proteomic studies indicate that many key responses and signal transduction events are rapidly activated at the initial flooding stress in soybean. To explore the flooding-tolerance mechanisms of soybean, particularly at the initial flooding stress, flooding-tolerant mutant and ABA-treated soybeans were used as materials. Because they exhibited the flooding tolerance, the commonly changed proteins in plants at initial flooding stress are the marker proteins for flooding tolerance. To identify the marker proteins of flooding stress, gel-free/label-free proteomic technique was used. Furthermore, to determine the function of the identified proteins related to flooding tolerance, bioinformatic, transcriptomic, enzymatic, and physiological analyses were performed.
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Mass Spectrometry Analysis
Peptides in 0.1% formic acid were loaded onto an Ultimate 3000 nano liquid chromatography (LC) system (Dionex, Germering, Germany) equipped with a C18 PepMap trap column (300 μm ID × 5 mm, Dionex). The peptides were eluted from the trap column and separated 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, Nikkyo Technos, Tokyo, Japan) with a spray voltage of 1.8 kV. The peptide ions in the spray were analyzed on a nanospray LTQ XL Orbitrap mass spectrometry (MS) (Thermo Fisher Scientific, San Jose, CA, USA) operated in data-dependent acquisition mode with the installed Xcalibur software (version 2.0.7, Thermo Fisher Scientific). Full-scan mass spectra were acquired in the MS over 400−1500 m/z with a resolution of 30 000. A lock mass function was used to obtain high mass accuracy.17 The three most intense precursor ions above a threshold of 500 were selected for collision-induced fragmentation in the linear ion trap at a normalized collision energy of 35% after accumulation to a target value of 1000. Dynamic exclusion was employed within 90 s to prevent the repetitive selection of peptides. Acquired spectra were used for protein identification.
EXPERIMENTAL PROCEDURES
Plant Materials and Treatments
Seeds of soybean (Glycine max L. cultivar Enrei) were sterilized with 1% sodium hypochlorite solution and rinsed in water. For initial flooding stress, seeds were sown on silica sand with water in a plastic case (180 × 140 × 45 mm3), and 2-day-old soybeans were treated with water for 3 h. For survival stages from flooding stress, seeds were sown on silica sand wetted with water in a plastic case (150 × 60 × 100 mm3), and 2-dayold soybeans were treated with water for 1, 2, 3, and 4 days. Soybeans were grown in a growth chamber illuminated with white fluorescent light (160 μmol m−2 s−1, 16 h light period/ day) at 25 °C. For ABA treatment, 10 μM ABA was supplied at the same time with flooding stress.10 As mutant line, flooding tolerant mutant9 was used after backcross with wild type soybean. Root and cotyledon were collected as samples. Three independent experiments were performed as biological replicates for all experiments. Biological replicate means that the plants were sown on the different days. For each treatment point, 10 plants were used.
Protein Identification using Acquired Mass Spectrometry Data
Protein identification was performed using the Mascot search engine (version 2.5.1, Matrix Science, London, UK) and a soybean peptide database (54,175 sequences) obtained from the Phytozome-Glycine max database (version 9.1, http://www. phytozome.net/soybean).18 DTA files were generated from acquired raw data files and then converted to Mascot generic files using Proteome Discoverer software (version 1.4.0.288, Thermo Fisher Scientific). The parameters used in the Mascot searches were as 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 precursor tolerance was set at 10 ppm, fragment mass tolerance was set at 0.8 Da, and peptide charges were set at +2, + 3, and +4. Peptide cutoff score was 10, and for peak filtration the S/N threshold (FT-only) was set at 1.5. An automatic decoy database search was performed as part of the search. Search results were filtered with the Mascot percolator to improve the accuracy and sensitivity of peptide identification.19 False discovery rates (false positive/(false postive + true postive)) for peptide identification of all searches were less than 1.0%. Peptides with a percolator ion score of more than 13 (p < 0.05) were used for protein identification. Search results were exported in XML format for comparison analysis.
Extraction of Proteins
A portion (0.5 g) of samples was ground to powder in liquid nitrogen using a mortar and pestle. Proteins were extracted as described by Komatsu et al.10 The final pellet of crude protein extract was dried and resuspended by vortexing for 60 min at 25 °C in lysis buffer consisting of 8 M urea, 2 M thiourea, 5% CHAPS, and 2 mM tributylphosphine. The suspension was then centrifuged at 20 000g for 20 min at 25 °C, and the supernatant was collected as crude protein extract. The protein concentration was determined using the Bradford method16 with bovine serum albumin as the standard. Purification and Digestion of Proteins
Proteins (150 μg) were purified by phase separation in the organic layer. After the volume was adjusted to 150 μL, 600 μL of methanol was added, and the solution was thoroughly mixed. Subsequently, 150 μL of chloroform was added, and 400 μL of water was added to the solution. The mixture was vortexed and centrifuged at 20 000g for 5 min. The upper aqueous phase was discarded, 400 μL of methanol was added to the organic phase, and samples were centrifuged at 20 000g for 5 min. The pellets were dried and resuspended in 50 mM NH4HCO3. Proteins were reduced with 50 mM dithiothreitol for 30 min at 56 °C, alkylated with 50 mM iodoacetamide for 30 min at 37 °C in
Differential Analysis of the Identified Proteins
To compare protein content between samples, extracted ion chromatograms were generated based on a comparison approach using SIEVE software (version 2.1.377, Thermo Fisher Scientific). The chromatographic peaks detected by MS were aligned, and the peptide peaks were detected as a frame B
DOI: 10.1021/acs.jproteome.6b00196 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research
less than 0.05 was considered as statistical significance. All calculations were performed using SPSS software (version 12.0J, IBM, Armonk, NY, USA).
using the following settings: frame time width (5 min); frame m/z width (10 ppm); and produce frames on all parent ions scanned by MS/MS. Chromatographic peak areas of each sample within a single frame were compared, and the ratios between samples in each frame were determined. The frames detected in the MS/MS scan were matched to the imported Mascot search results. The ratio of peptides between samples was determined from the variance-weighted average of the ratios in frames, which matched the peptides in the MS/MS spectrum. The ratios of peptides were further integrated to determine the ratio of the corresponding protein. In the differential analysis of protein abundance, total ion current was used for normalization. The minimum requirement for the identification of a protein was a minimum of two matched peptides. The outliers of ratio were deleted in the frames table filter according to frame area. The isoforms were manually removed based on protein ID. Significant changes in the abundance of proteins between samples were analyzed (p < 0.05). Three biological replicates were used for each comparison in this study.
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RESULTS
Identification of Flooding Stress-Responsive Proteins in Flooding-Tolerant and ABA-Treated Soybeans
To investigate the initial responses of soybean to flooding, 2day-old plants of flooding-tolerant mutant9 and soybean treated with 10 μM ABA, which also exhibits flooding tolerance,10 were exposed to flooding stress for 3 h. To identify specific proteins that were changed in the mutant and ABA-treated plants in response to flooding stress, proteomic technique was used. Proteins extracted from the roots and cotyledons were analyzed using nanoLC−MS/MS (Figure 1), and protein abundance was
RNA Extraction and Quantitative Reverse Transcription Polymerase Chain Reaction Analysis
A portion (0.1 g) of samples was ground in liquid nitrogen using a mortar/pestle, and total RNA was extracted using RNeasy Plant Mini kit (Qiagen, Valencia, CA, USA). RNA was reverse-transcribed using iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed in a 10 μL of reaction using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and a MyiQ single-color realtime PCR detection system (Bio-Rad). The PCR conditions were as follows: 95 °C for 30 s, followed by 45 cycles of 95 °C for 10 s and 60 °C for 30 s. Gene expression was normalized using 18S rRNA as an internal control. The primers were designed using the Primer3 Plus web interface (http://frodo.wi. mit.edu) (Table S1). Specificity of primers was confirmed by BLASTN search against the Phytozome-Glycine max database with the designed primers as queries by melt curve analysis. The sizes of the PCR amplified products were analyzed using agarose gel electrophoresis (Figure S3). Analysis of Protein Function
Protein function was predicted and categorized using MapMan bin codes.20 Pathway mapping of identified proteins was visualized using MapMan bin software (Version 3.6.0RC1).21 Cluster analysis was performed using Genesis software (version 1.7.6) (https://genome.tugraz.at/).22
Figure 1. Experimental design for the proteomic analysis of a floodingtolerant mutant and ABA-treated soybeans. For initial flooding stress, 2-day-old soybeans were flooded for 3 h. For continuous flooding stress, 2-day-old soybeans were flooded for 1, 2, 3, and 4 days. For ABA treatment, 10 μM ABA was added to the water used for flooding. The mutant line was only exposed to flooding stress. Roots and cotyledons were collected as samples. Proteins were extracted from the collected tissues, digested, and analyzed using nanoLC−MS/MS. Three independent experiments were performed as biological replicates for all experiments.
Analysis of Lignification
Root sections (0.1 mm) were manually sliced at 10 mm from root tip. The root sections were covered with mineral oil and observed using fluorescence microscope (Axioscope; Zeiss, Jena, Germany), which equipped with epifluorescence illumination with Mercury lamp (HBO 50W). The filter set, which used to observe autofluorescence of lignin, consisted of a bandpass DAPI filter 340−380 nm (exciter). Pictures were taken with a digital camera (DSC-S85; Sony, Tokyo, Japan) mounted on the microscope.23
compared with the flooding-stressed wild-type soybean. A total of 1045 (Table S2) and 384 proteins (Table S3) were significantly changed in abundance in the roots of mutant and ABA-treated soybeans, respectively. Among these identified proteins, 146 proteins were commonly changed between the mutant and ABA-treated soybean (Table 1). In addition, 500 (Table S4) and 319 proteins (Table S5) were significantly changed in abundance in the cotyledons of mutant and ABA-
Statistical Analysis
The statistical significance of results was evaluated with the Student’s t-test for only two groups and the one-way ANOVA test with Tukey’s comparison for multiple groups. A p-value of C
DOI: 10.1021/acs.jproteome.6b00196 J. Proteome Res. XXXX, XXX, XXX−XXX
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Journal of Proteome Research
Table 1. Commonly Changed Proteins in the Roots of Flooding-Tolerant Mutant and ABA-Treated Soybean Seedlings during the Initial Stages of Flooding Stress mutant line protein IDa Glyma06g14360.1 Glyma04g40470.1 Glyma04g40430.2 Glyma15g18170.3 Glyma12g17181.1 Glyma09g06860.1 Glyma08g23750.3 Glyma07g02270.1 Glyma15g42620.1 Glyma08g16130.1 Glyma08g15000.1 Glyma08g14995.1 Glyma05g31760.2 Glyma13g19930.1 Glyma10g05580.1 Glyma03g33460.1 Glyma08g09200.3 Glyma05g26290.1 Glyma12g36940.2 Glyma10g36780.1 Glyma15g02610.1 Glyma13g42830.1 Glyma07g01540.1 Glyma15g17010.1 Glyma05g26320.1 Glyma01g40150.1 Glyma19g37370.1 Glyma13g21520.1 Glyma10g07680.1 Glyma03g34700.1 Glyma10g02270.1 Glyma02g02140.1 Glyma19g39240.1 Glyma03g36560.1 Glyma15g10220.1 Glyma14g03380.1 Glyma13g28831.1 Glyma18g01110.1 Glyma16g28550.2 Glyma11g37160.1 Glyma08g10910.1 Glyma05g27940.1 Glyma16g13580.1 Glyma09g02790.1 Glyma16g17370.4
ABA-treated soybean
description
MPb
ratioc
SD
p-value
MPb
ratioc
SD
p-value
Ribosomal protein L6 family Ribosomal protein L6 family Ribosomal protein L6 family Ribosomal protein L30/L7 family protein Ribosomal protein L30/L7 family protein Ribosomal protein L30/L7 family protein Ribosomal protein L30/L7 family protein Ribosomal protein L30/L7 family protein Ribosomal protein L6 family protein Ribosomal protein L6 family protein Ribosomal protein L6 family protein Ribosomal protein L6 family protein Ribosomal protein L6 family protein Ribosomal protein L4/L1 family Ribosomal protein L4/L1 family Ribosomal protein L4/L1 family Ribosomal protein L35Ae family protein Ribosomal protein L35Ae family protein R protein L3 B ribosomal protein 1 Ribosomal L28e protein family Ribosomal L28e protein family Ribosomal L28e protein family Ribosomal L27e protein family Ribosomal L27e protein family Translation protein SH3_like family protein ribosomal protein L24 ribosomal protein L24 ribosomal protein L24 ribosomal protein L24 Ribosomal L22e protein family Ribosomal L22e protein family Translation protein SH3_like family protein Translation protein SH3-like family protein Ribosomal protein L18ae/LX family protein Ribosomal protein L18ae/LX family protein Ribosomal protein L18ae/LX family protein Ribosomal protein L22p/L17e family protein Ribosomal protein L22p/L17e family protein Ribosomal protein L22p/L17e family protein Ribosomal protein L22p/L17e family protein Ribosomal protein L22p/L17e family protein Ribosomal protein L13 family protein Ribosomal protein L13 family protein breast basic conserved 1
5 6 5 6
0.191 0.128 0.130 0.086
0.347 0.324 0.357 0.286
0.000 0.000 0.002 0.000
7 6 3 7
0.546 0.569 0.481 0.270
0.298 0.366 0.433 0.294
0.028 0.051 0.027 0.000
protein protein protein protein
2
0.171
0.634
0.023
2
0.479
0.662
0.054
protein synthesis
5
0.075
0.308
0.000
6
0.270
0.302
0.000
protein synthesis
6
0.103
0.353
0.000
6
0.481
0.360
0.005
protein synthesis
7
0.096
0.333
0.000
7
0.489
0.333
0.004
protein synthesis
6 6 7 7 6 12 10 3 2
0.191 0.142 0.125 0.209 0.328 0.525 0.129 0.137 0.150
0.352 0.348 0.335 0.230 0.334 0.126 0.178 0.559 0.900
0.001 0.001 0.000 0.001 0.002 0.000 0.000 0.007 0.023
6 6 7 7 7 11 11 3 2
0.317 0.350 0.327 0.499 0.437 0.430 0.440 0.470 0.268
0.337 0.334 0.336 0.303 0.273 0.180 0.186 0.496 0.598
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.017 0.001
protein protein protein protein protein protein protein protein protein
3
0.125
0.546
0.000
2
0.346
0.581
0.004
protein synthesis
3 10 3 3 3 2 2 2
0.150 0.190 0.315 0.315 0.299 0.317 0.089 0.043
0.232 0.174 0.517 0.517 0.435 0.267 0.469 0.939
0.000 0.000 0.001 0.001 0.001 0.006 0.004 0.002
3 10 3 3 3 3 3 2
0.547 0.645 0.337 0.337 0.452 0.442 0.459 0.459
0.329 0.209 0.497 0.497 0.401 0.532 0.574 0.520
0.017 0.050 0.001 0.001 0.002 0.018 0.040 0.019
protein protein protein protein protein protein protein protein
synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis
3 3 3 3 3 3 2
0.111 0.111 0.111 0.111 0.448 0.448 0.075
0.463 0.463 0.463 0.463 0.270 0.270 0.447
0.000 0.000 0.000 0.000 0.000 0.000 0.001
2 2 2 2 4 4 2
1.576 1.576 1.576 1.576 0.574 0.574 0.395
0.420 0.420 0.420 0.420 0.383 0.383 0.536
0.053 0.053 0.053 0.053 0.054 0.054 0.011
protein protein protein protein protein protein protein
synthesis synthesis synthesis synthesis synthesis synthesis synthesis
2
0.075
0.447
0.001
2
0.395
0.536
0.011
protein synthesis
2
0.091
0.451
0.003
3
0.284
0.634
0.037
protein synthesis
2
0.091
0.451
0.003
3
0.284
0.634
0.037
protein synthesis
3
0.108
0.386
0.000
4
0.290
0.445
0.014
protein synthesis
3
0.193
0.406
0.006
3
0.441
0.588
0.033
protein synthesis
3
0.193
0.406
0.006
3
0.441
0.588
0.033
protein synthesis
4
0.191
0.222
0.001
4
0.351
0.416
0.001
protein synthesis
3
0.193
0.406
0.006
3
0.441
0.588
0.033
protein synthesis
3
0.193
0.406
0.006
3
0.441
0.588
0.033
protein synthesis
2 4 5
0.251 0.230 0.111
0.273 0.252 0.251
0.000 0.000 0.000
3 5 6
0.553 0.590 0.606
0.388 0.325 0.301
0.032 0.047 0.045
protein synthesis protein synthesis protein synthesis
D
functiond synthesis synthesis synthesis synthesis
synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis
DOI: 10.1021/acs.jproteome.6b00196 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research Table 1. continued mutant line protein ID
a
Glyma09g34760.5 Glyma04g14640.3 Glyma15g36190.2 Glyma12g15800.1 Glyma0985s00200.1 Glyma06g48130.1 Glyma06g47510.1 Glyma04g12320.2 Glyma03g21710.1 Glyma19g06730.1 Glyma13g07650.1 Glyma15g08210.1 Glyma13g31130.1 Glyma13g24630.1 Glyma08g45770.1 Glyma07g31840.1 Glyma17g22200.1 Glyma17g22161.1 Glyma16g23730.1 Glyma14g06170.1 Glyma03g06440.1 Glyma02g43080.1 Glyma02g05370.1 Glyma01g31270.1 Glyma20g28780.1 Glyma11g00890.1 Glyma10g39050.1 Glyma01g44700.1 Glyma12g04510.2 Glyma11g12300.1 Glyma06g01310.1 Glyma19g43190.1 Glyma11g02250.1 Glyma11g02190.1 Glyma05g34570.1 Glyma02g09370.1 Glyma20g30730.1 Glyma02g08690.1 Glyma20g02170.1 Glyma11g32827.1 Glyma07g34440.1 Glyma08g05290.1 Glyma07g33800.1 Glyma05g34350.1 Glyma02g11540.1 Glyma15g10210.1 Glyma13g28840.1 Glyma17g14370.1 Glyma05g03850.2 Glyma17g11430.1
description breast basic conserved 1 breast basic conserved 1 Ribosomal L5P family protein Ribosomal L5P family protein Ribosomal L5P family protein Ribosomal L5P family protein Ribosomal L5P family protein Ribosomal L5P family protein Ribosomal protein S8e family protein Ribosomal protein S7e family protein Ribosomal protein S7e family protein Ribosomal protein S6e Ribosomal protein S6e Ribosomal protein S6e Ribosomal protein S6e Ribosomal protein S6e Ribosomal protein S4 (RPS4A) family protein Ribosomal protein S4 (RPS4A) family protein Ribosomal protein S4 (RPS4A) family protein Ribosomal protein S4 (RPS4A) family protein Ribosomal protein S4 (RPS4A) family protein Ribosomal protein S4 (RPS4A) family protein Ribosomal protein S4 (RPS4A) family protein Ribosomal protein S4 (RPS4A) family protein Ribosomal protein S3Ae Ribosomal protein S3Ae Ribosomal protein S3Ae Ribosomal protein S3Ae Ribosomal protein S25 family protein Ribosomal protein S25 family protein Ribosomal protein S25 family protein Ribosomal protein S24e family protein Ribosomal protein S5 family protein Ribosomal protein S5 family protein Ribosomal protein S5 family protein Ribosomal protein S5 family protein S18 ribosomal protein S18 ribosomal protein Ribosomal S17 family protein Ribosomal S17 family protein Ribosomal S17 family protein Ribosomal protein S5 domain 2-like superfamily protein Ribosomal protein S5 domain 2-like superfamily protein Ribosomal protein S5 domain 2-like superfamily protein Ribosomal protein S5 domain 2-like superfamily protein Ribosomal protein S11 family protein Ribosomal protein S11 family protein ribosomal protein S13A ribosomal protein S13A ribosomal protein S11_beta
b
MP
ratio
c
ABA-treated soybean
SD
p-value
MP
b
ratioc
SD
p-value
functiond
7 7 2 2 2 2 2 2 5 3 3 3 4 3 4 3 3
0.229 0.248 0.153 0.153 0.153 0.153 0.153 0.153 0.122 0.112 0.112 0.068 0.084 0.097 0.096 0.097 0.109
0.209 0.205 0.544 0.544 0.544 0.544 0.544 0.544 0.361 0.365 0.365 0.536 0.333 0.489 0.320 0.489 0.406
0.000 0.000 0.008 0.008 0.008 0.008 0.008 0.008 0.000 0.000 0.000 0.001 0.000 0.001 0.000 0.001 0.001
7 7 2 2 2 2 2 2 7 4 5 2 3 3 4 3 3
0.599 0.613 0.465 0.465 0.465 0.465 0.465 0.465 0.579 0.316 0.392 0.314 0.383 0.342 0.400 0.342 0.348
0.263 0.271 0.551 0.551 0.551 0.551 0.551 0.551 0.310 0.404 0.356 0.329 0.292 0.312 0.280 0.312 0.386
0.034 0.041 0.037 0.037 0.037 0.037 0.037 0.037 0.049 0.000 0.000 0.006 0.008 0.008 0.010 0.008 0.000
protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein
2
0.141
0.481
0.002
2
0.353
0.445
0.001
protein synthesis
8
0.078
0.246
0.000
8
0.358
0.243
0.000
protein synthesis
8
0.078
0.246
0.000
8
0.358
0.243
0.000
protein synthesis
6
0.087
0.277
0.000
6
0.367
0.273
0.000
protein synthesis
8
0.084
0.258
0.000
8
0.367
0.236
0.000
protein synthesis
8
0.078
0.246
0.000
8
0.358
0.243
0.000
protein synthesis
6
0.087
0.277
0.000
6
0.367
0.273
0.000
protein synthesis
8 6 7 6 3 3 3 3
0.115 0.102 0.116 0.111 0.275 0.275 0.285 0.057
0.226 0.277 0.227 0.244 0.506 0.506 0.518 0.768
0.000 0.000 0.000 0.000 0.003 0.003 0.004 0.000
7 5 7 6 3 3 3 3
0.468 0.352 0.468 0.417 0.274 0.274 0.283 0.392
0.297 0.324 0.297 0.288 0.411 0.411 0.413 0.784
0.001 0.000 0.001 0.000 0.000 0.000 0.000 0.046
protein protein protein protein protein protein protein protein
synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis
7 7 6 7 4 3 5 3 5 2
0.048 0.048 0.047 0.048 0.193 0.150 0.492 0.688 0.492 0.115
0.392 0.392 0.421 0.392 0.190 0.237 0.298 0.424 0.298 0.431
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.026 0.000 0.000
7 7 7 7 4 3 4 3 4 2
0.334 0.334 0.587 0.334 0.457 0.385 0.660 0.667 0.660 0.391
0.288 0.288 0.203 0.288 0.523 0.674 0.196 0.204 0.196 0.631
0.000 0.000 0.000 0.000 0.049 0.050 0.021 0.032 0.021 0.018
protein protein protein protein protein protein protein protein protein protein
synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis
3
0.132
0.388
0.000
3
0.483
0.486
0.017
protein synthesis
2
0.115
0.431
0.000
2
0.391
0.631
0.018
protein synthesis
3
0.132
0.388
0.000
3
0.483
0.486
0.017
protein synthesis
5 6 4 4 2
0.236 0.239 0.177 0.177 0.356
0.261 0.255 0.183 0.183 0.427
0.000 0.000 0.000 0.000 0.029
5 6 4 4 2
0.514 0.517 0.426 0.426 0.178
0.248 0.237 0.375 0.375 0.598
0.001 0.001 0.007 0.007 0.001
protein protein protein protein protein
E
synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis
synthesis synthesis synthesis synthesis synthesis
DOI: 10.1021/acs.jproteome.6b00196 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research Table 1. continued mutant line protein ID
a
Glyma13g23400.1 Glyma19g30550.1
description
b
MP
ratio
c
ABA-treated soybean
SD
p-value
MP
b
ratioc
SD
p-value
functiond
2 7
0.356 2.007
0.427 0.196
0.029 0.015
2 7
0.178 1.455
0.598 0.114
0.001 0.001
protein synthesis protein synthesis
7
2.188
0.179
0.005
7
1.480
0.125
0.003
protein synthesis
6
2.218
0.194
0.011
6
1.588
0.164
0.001
protein synthesis
6
1.811
0.230
0.041
6
1.452
0.125
0.001
protein synthesis
6 7 4
3.088 3.077 1.915
0.396 0.365 0.417
0.033 0.022 0.050
6 7 4
2.041 2.046 1.567
0.234 0.214 0.236
0.001 0.000 0.027
protein folding protein folding RNA−RNA binding
3
0.111
0.613
0.009
3
0.386
0.402
0.007
RNA−RNA binding
7 4 6 3
4.702 2.370 3.454 0.111
0.327 0.264 0.330 0.613
0.004 0.016 0.022 0.009
5 4 4 3
2.802 1.651 2.291 0.386
0.297 0.176 0.328 0.402
0.001 0.004 0.004 0.007
RNA−RNA RNA−RNA RNA−RNA RNA−RNA
4
2.277
0.357
0.030
5
1.638
0.235
0.008
RNA−RNA binding
4 3
1.990 1.762
0.320 0.269
0.024 0.044
4 3
1.650 1.612
0.244 0.271
0.011 0.034
RNA−RNA binding RNA−RNA binding
4
3.069
0.376
0.020
3
1.584
0.311
0.038
4
3.347
0.364
0.038
3
1.666
0.249
0.005
5
2.908
0.355
0.033
6
1.697
0.203
0.001
Glyma12g03470.1
ribosomal protein S11_beta Nascent polypeptide_associated complex (NAC)_ alpha subunit Nascent polypeptide_associated complex (NAC)_ alpha subunit Nascent polypeptide_associated complex (NAC)_ alpha subunit Nascent polypeptide_associated complex (NAC)_ alpha subunit chaperonin 20 chaperonin 20 RNA_binding (RRM/RBD/RNP motifs) THO complex subunit 4-like isoform X1 KH domain_containing protein glycine_rich RNA_binding protein 3 KH domain_containing protein THO complex subunit 4-like isoform X1 RNA_binding (RRM/RBD/RNP motifs) glycine_rich RNA_binding protein 3 cold_ circadian rhythm RNA binding 2 Eukaryotic aspartyl protease family protein Eukaryotic aspartyl protease family protein Eukaryotic aspartyl protease family protein glycine_rich protein 2B
3
1.879
0.135
0.002
3
1.651
0.256
0.025
Glyma11g11290.5
glycine_rich protein 2B
3
1.873
0.133
0.001
3
1.616
0.249
0.025
Glyma20g23080.1 Glyma12g00800.1 Glyma10g28890.2 Glyma09g36560.1 Glyma17g17730.1 Glyma16g27900.1 Glyma09g02600.1 Glyma05g22180.1 Glyma15g00900.1
11 5 15 6 9 8 3 8 5
1.814 0.254 1.864 0.244 4.359 5.365 3.132 4.336 1.647
0.171 0.170 0.140 0.158 0.195 0.277 0.471 0.214 0.333
0.001 0.000 0.000 0.000 0.001 0.001 0.039 0.002 0.053
11 5 13 7 7 7 4 6 5
1.554 0.636 1.555 0.648 1.737 1.747 2.701 1.709 1.731
0.144 0.267 0.131 0.226 0.231 0.228 0.309 0.259 0.255
0.001 0.013 0.001 0.043 0.007 0.020 0.018 0.015 0.018
5
1.647
0.333
0.053
5
1.731
0.255
0.018
Glyma14g35430.1
calreticulin 1b BCL_2_associated athanogene 7 calreticulin 1b BCL_2_associated athanogene 7 root hair specific 19 Peroxidase superfamily protein Peroxidase superfamily protein root hair specific 19 NADH_ubiquinone oxidoreductase 24 kDa subunit NADH_ubiquinone oxidoreductase 24 kDa subunit cytochrome C oxidase 6B
3
3.893
0.450
0.033
3
3.255
0.267
0.008
Glyma06g39800.1 Glyma08g06950.1 Glyma07g30290.1 Glyma17g06170.1
Adenine nucleotide alpha hydrolases mitochondrial HSO70 2 mitochondrial HSO70 2 nucleosome assembly protein 1;2
5 13 13 7
1.641 1.426 1.496 2.484
0.285 0.132 0.132 0.234
0.052 0.028 0.015 0.008
5 13 13 7
1.653 1.258 1.280 1.490
0.262 0.099 0.098 0.165
0.007 0.050 0.023 0.006
Glyma13g16500.1
nucleosome assembly protein 1;2
7
2.478
0.235
0.008
6
1.457
0.172
0.013
Glyma03g34680.1
late embryogenesis abundant protein_ putative
2
0.007
1.122
0.016
2
1.576
0.594
0.000
Glyma03g40280.1
copper/zinc superoxide dismutase 1
2
2.089
0.133
0.000
2
1.660
0.252
0.018
Glyma12g03210.3
cell elongation protein/DWARF1/ DIMINUTO (DIM) cell elongation protein/DWARF1/ DIMINUTO (DIM)
5
0.347
0.359
0.000
4
0.743
0.338
0.028
RNA regulation of transcription RNA regulation of transcription RNA regulation of transcription RNA regulation of transcription RNA regulation of transcription signaling calcium signaling calcium signaling calcium signaling calcium misc. peroxidases misc. peroxidases misc. peroxidases misc. peroxidases mitochondrial electron transport mitochondrial electron transport mitochondrial electron transport stress abiotic stress abiotic heat stress abiotic heat DNA synthesis/ chromatin structure DNA synthesis/ chromatin structure development late embryogenesis abundant redox dismutases and catalases hormone metabolism
5
0.347
0.359
0.000
4
0.743
0.338
0.028
hormone metabolism
Glyma19g30540.1 Glyma03g27580.1 Glyma03g27570.1 Glyma15g19970.1 Glyma09g08340.1 Glyma20g34330.1 Glyma18g04530.2 Glyma17g34850.1 Glyma17g08630.1 Glyma14g10670.1 Glyma11g33670.1 Glyma06g33940.2 Glyma05g00400.1 Glyma04g01421.1 Glyma13g26600.1 Glyma11g25650.1 Glyma04g17600.1
Glyma13g44380.1
Glyma11g11020.3
F
binding binding binding binding
DOI: 10.1021/acs.jproteome.6b00196 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research Table 1. continued mutant line protein ID
a
description
b
MP
ratio
c
ABA-treated soybean
SD
p-value
MP
b
ratioc
SD
p-value
functiond
Glyma13g33590.1
glyoxalase II 3
6
1.545
0.208
0.041
5
0.959
0.129
0.003
Glyma12g08470.1
lactoylglutathione lyase protein/ glyoxalase I family protein lactoylglutathione lyase protein/ glyoxalase I family protein Kinase_related protein of unknown function (DUF1296) fruit protein pKIWI501-like selenium binding uncharacterized protein LOC100783793 gelsolin-related protein of 125 kDalike Late embryogenesis abundant protein (LEA) family protein Lipase/lipooxygenase_ PLAT/LH2 family protein
6
3.170
0.388
0.048
3
1.743
0.356
0.052
biodegradation of xenobiotics amino acid metabolism
6
3.170
0.388
0.048
3
1.743
0.356
0.052
amino acid metabolism
6
2.366
0.291
0.029
2
1.954
0.218
0.003
not assigned
3 2 4
5.178 0.262 2.817
0.299 0.164 0.200
0.003 0.001 0.001
3 3 4
4.526 0.637 4.525
0.403 0.555 0.284
0.010 0.015 0.000
not assigned not assigned not assigned
5
2.969
0.257
0.011
4
3.227
0.271
0.001
not assigned
4
0.147
0.465
0.000
4
0.275
0.456
0.000
not assigned
3
1.891
0.274
0.039
2
1.474
0.279
0.036
not assigned
Glyma11g20000.1 Glyma15g13211.1 Glyma15g12170.1 Glyma14g17190.1 Glyma09g01320.1 Glyma07g39540.1 Glyma13g31120.1 Glyma08g14550.1 a
Protein ID is according to the Phytozome database. bMP means matched peptides. cRatio is calculated using treatments and control. dFunctional category is obtained from MapMan bin codes.
treated soybeans, respectively. Among these flooding-responsive cotyledon proteins, 217 proteins were commonly changed between the mutant and ABA-treated soybeans (Table S6). To better understand the function of the identified proteins in soybean at the initial stages of flooding stress, functional categorization was performed using MapMan bin codes (Figure 2). The significantly changed proteins in the mutant were categorized as protein synthesis, signaling, cell, amino acid metabolism, transport, and stress-related proteins, whereas the identified proteins in ABA-treated soybean were predominantly related to protein synthesis, RNA regulation, redox, development, and stress. The commonly changed flooding-responsive proteins between the mutant and ABA-treated plants were categorized as protein synthesis and RNA regulation proteins and exhibited similar changes in abundance between mutant and ABA-treated soybean (Figure 2). Among the protein synthesis-related proteins, many ribosomal proteins were identified and decreased in response to flooding stress, with the exception of nascent polypeptide associated complex (NAC) and chaperonin 20 (Table 1). In the cotyledons of the flooding-tolerant mutant and ABA-treated soybeans, protein synthesis and stress-related proteins were commonly changed; however, they were increased in mutant line and decreased in ABA-treated soybean (Figure S1). These comparative proteomic results indicated that protein synthesisand RNA regulation-related proteins were involved at the initial flooding stress in the mutant and ABA-treated soybeans.
involved several pathways including cell wall, tricarboxylic acid cycle, oxidative pentose phosphate, photorespiration, and reactive oxygen species scavenging system that were increased in the mutant line, whereas they were inhibited at the initial flooding stress in the ABA-treated soybean (Figure S2). Organ-Specific Expression of Genes Encoding Proteins Related to Protein Synthesis and RNA Regulation
A number of protein synthesis- and RNA regulation-related proteins were significantly and commonly changed in abundance in the roots of the flooding-tolerant mutant and ABA-treated soybeans (Figure 2). To confirm the functions of these proteins at the initial flooding stress, the specific gene expression of these proteins in the roots and cotyledons was analyzed by qRT-PCR. The sizes of the PCR products amplified were the same as those predicated based on the designed primers (Figure S3). Among the protein synthesisrelated functional categories, most of the analyzed proteins were decreased in abundance with the exception of NAC and chaperonin 20. Consistent with this increase at the protein abundance, the mRNA expression level of NAC and chaperonin 20 was up-regulated in the roots of both the mutant and ABA-treated soybeans compared to that in the wild-type soybean at the initial flooding stress. In the cotyledons, the mRNA expression level of chaperonin 20 was up-regulated in the mutant in response to flooding stress (Figure 4). Among seven RNA regulation-related proteins, the mRNA expression level of THO complex subunit 4, glycine-rich RNAbinding protein 3, and eukaryotic aspartyl protease family protein was up-regulated in the root of both the mutant and ABA-treated soybeans at the initial flooding stress, whereas the mRNA expression level of KH domain-containing protein and glycine-rich protein 2B was up-regulated in the root of only the mutant line. The mRNA expression level of RNA-binding (RRM/RBD/RNP motifs) and cold circadian rhythm/RNA binding 2 did not change. In addition, the mRNA expression profiles of these genes were in accordance with the patterns of protein abundance except for THO complex subunit 4 (Figure 5). In the cotyledons, the mRNA expression level of KH domain containing protein, glycine-rich RNA binding protein 3,
Primary Metabolic Pathways Affected at the Initial Stage of Flooding Stress
To determine the soybean metabolic pathways involved in the initial flooding tolerance, the functional visualization of significantly changed proteins was performed using MapMan software (Figure 3, Figure S2). Mapping of the identified flooding-responsive proteins from the mutant and ABA-treated soybeans on the known metabolic pathways indicated that cell wall, fermentation, minor CHO metabolism, C1 metabolism, and nucleotide metabolism were clearly activated in the roots of both plants. The secondary and amino acid metabolic pathways were also specifically activated in the roots of the floodingtolerant mutant (Figure 3). In the cotyledons, the proteins G
DOI: 10.1021/acs.jproteome.6b00196 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research
flooding. For mutant line, only flooding stress was applied. Proteins were extracted from the roots and analyzed using nanoLC−MS/MS (Figure 1). To identify specific proteins changed in mutant and ABA-treated soybeans under flooding for 2 days, protein abundance was compared with floodingstressed wild-type soybean. A total of 1403 proteins (Table S7) and 131 proteins (Table S8) were significantly changed in the mutant line and ABA-treated soybean, respectively, compared to wild-type soybean in response to 2-day flooding exposure. Among the changed proteins, 78 proteins were commonly changed between the mutant and ABA-treated soybeans (Table 2). Functional categorization of the identified flooding-responsive proteins using MapMan bin codes (Figure 6) revealed that proteins in the mutant were predominantly categorized as protein synthesis, amino acid metabolism, cell, stress, and cell wall related proteins, whereas in the roots of ABA-treated soybean, proteins related to synthesis, cell, cell wall, redox, and development were changed in abundance. Proteins categorized as protein, cell wall, cell, and redox homeostasis related proteins were commonly changed in the flooding-tolerant mutant and ABA-treated plants. Most of the differentially changed proteins were increased in abundance and exhibited similar profiles in the mutant and ABA-treated soybeans (Figure 6). Temporal Changes in Flooding-Responsive Proteins in the Roots of Mutant and ABA-Treated Soybeans
To examine whether the flooding-responsive proteins that were commonly identified in the mutant and ABA-treated soybeans contribute to the survival of soybean under flooding conditions, proteins were extracted from the roots of 2-day-old soybeans flooded for 1, 2, 3, and 4 days and then analyzed using nanoLC−MS/MS (Figure 1). Time-dependent changes in protein level were examined using SIEVE software. A total of 48 proteins (Table S9) and 57 proteins (Table S10) were significantly changed in the mutant and ABA-treated soybean, respectively, in response to flooding stress compared to 2-dayold soybeans. Among the identified proteins, five proteins were commonly changed between the mutant and ABA-treated soybeans under flooding stress (Table 3). To further explore the function of the differentially changed proteins, functional categorization was performed using MapMan bin codes (Figure 7). The significantly changed proteins in the mutant line were predominantly categorized as protein synthesis, fermentation, major CHO metabolism, and cell wall proteins, whereas in the ABA-treated soybean, the identified proteins were related to protein synthesis, cell wall, development, and RNA regulation (Figure 7). In addition, cluster analysis clearly indicated that the protein abundance ratios of uricase/nodulin 35, aluminum-induced protein with YGL/ LRDR motifs, enolase, and polygalacturonase-inhibiting protein 1 were increased, whereas the ratio of RmlC-like cupin protein decreased in the roots of the flooding-stressed mutant and ABA-treated soybeans in a time-dependent manner (Figure 7). To further analyze changes in the temporal profile of the flooding-responsive proteins, the identified proteins were analyzed using a hierarchical clustering method. Forty-eight proteins in the flooding-tolerant mutant and 57 proteins in ABA-treated soybean were grouped into either Cluster I or II, which represent proteins that increased or decreased in abundance with time, respectively (Figure 8). In the mutant, Clusters I and II contained 27 and 21 proteins, respectively,
Figure 2. Functional distribution of proteins identified in the roots of the flooding-tolerant mutant and ABA-treated soybeans at the initial flooding stress. Two-day-old soybeans were flooded for 3 h, and proteins were extracted from the roots and analyzed using nanoLC− MS/MS. For ABA treatment, 10 μM ABA was added to the water used for flooding. For mutant, only flooding stress was applied. To identify specific flooding-responsive proteins in the mutant and ABA-treated soybean plants, protein abundances were compared with the floodingstressed wild-type and non-ABA treated soybean, respectively. Functional classification of the differentially abundant proteins was performed according to MapMan bin codes. A total of 1045 and 384 proteins from the mutant and ABA-treated soybean, respectively, were categorized based on functional distribution. A total of 146 common proteins were further categorized. Abbreviations: CHO, carbohydrates; ET, electron transport; TCA, tricarboxylic acid.
and cold circadian-rhythm RNA binding 2 was up-regulated in the mutant line, whereas the mRNA expression level of eukaryotic aspartyl protease family protein was up-regulated in ABA-treated soybean compared to the wild-type soybean under flooding stress. Notably, the mRNA expression level of these genes in the cotyledons was lower than that in the roots (Figure 5). Taken together, these results indicated that genes encoding protein synthesis- and RNA regulation-related proteins were specifically expressed in the roots of soybean at the initial flooding stress. Identification of Proteins Changed in Roots of Mutant and ABA-Treated Soybeans after 2-Day Flooding Stress
To further explore the flooding-response mechanisms in soybean, 2-day-old soybeans were flooded for 2 days. For ABA treatment, 10 μM ABA was applied at the same time with H
DOI: 10.1021/acs.jproteome.6b00196 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research
Figure 3. Mapping of proteins identified in the roots of the flooding-tolerant mutant and ABA-treated soybeans at the initial flooding stress. The 1045 and 384 flooding-responsive proteins identified in the mutant and ABA-treated soybean, respectively, at the initial flooding stress were mapped on known metabolic pathways of soybean using MapMan software. MapMan visualization showing changes in protein abundance based on the Log2FC of the differential protein ratio. Red and green colors indicate increased (Log2FC > 0) and decreased (Log2FC < 0) proteins, respectively.
whereas in ABA-treated soybean, Clusters I and II contained 7 and 50 proteins, respectively (Figure 8).
Protein synthesis-related proteins, including NAC and chaperonin 20, were significantly increased in abundance in the roots of the mutant and ABA-treated soybeans at initial flooding stress (Figure 4). However, they were increased and decreased in root of mutant and ABA-treated soybeans, respectively, during the survival stages under flooding (Figure S4). In addition, six RNA regulation-related proteins were also significantly increased in the roots of the mutant and ABAtreated soybeans at the initial flooding stress (Figure 5); however, these proteins decreased in abundance in the ABAtreated soybean under continuous flooding conditions. In roots of the flooding-tolerant mutant, KH domain-containing protein, eukaryotic aspartyl protease family protein, and cold circadian rhythm/RNA-binding 2 were increased after 4 days of flooding stress, and the protein abundance ratios of RNAbinding (RRM/RBD/RNP motifs) and glycine-rich protein 2B
Temporal Profiles of Nine Initial Flooding Stress Responsive Proteins in Following Flooding Condition
To confirm the function of initial stress responded nine proteins during the survival stages of soybean under flooding condition, a proteomic approach was applied. Among nine proteins, NAC, chaperonin 20, KH-domain containing protein, glycine-rich protein 2B, RNA binding (RRM/RBD/RNP motifs), glycine-rich RNA-binding protein 3, eukaryotic aspartyl protease family protein, and cold circadian rhythm/RNA binding 2 were identified; however, THO complex subunit 4 was not detected in either the flooding-tolerant mutant or ABAtreated soybean (Table S11). I
DOI: 10.1021/acs.jproteome.6b00196 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research
Figure 4. Organ-specific mRNA expression levels of genes related to protein synthesis in soybean under flooding stress. Two-day-old soybeans were flooded for 3 h, and total RNA was extracted from the roots and cotyledons of the flooding-tolerant mutant, wild-type, and ABA-treated soybean. For ABA treatment, 10 μM ABA was added to the water used for flooding. The mRNA expression level of NAC (Glyma03g27580.1) and chaperonin 20 (Glyma15g19970.1) was analyzed by qRT-PCR. Relative mRNA expression levels were normalized according to the abundance of 18S rRNA. Data are means ± SD from three independent biological replicates. The significance between the treated and control samples was analyzed using the Student’s t-test (∗, p-value
