Insights into the Response of Soybean Mitochondrial Proteins to Various Sizes of Aluminum Oxide Nanoparticles under Flooding Stress

Rapid developments in nanotechnology have led to the increasing use of nanoparticles (NPs) in the agricultural sector. For possible interactions between NPs and crops under flooding stress to be investigated, the molecular mechanisms in soybeans affected by exposure to various sizes of Al₂O₃ NPs were analyzed using a proteomic technique. In plants exposed to 30–60 nm Al₂O₃ NPs, the length of the root including hypocotyl was increased, and proteins related to glycolysis were suppressed. Exposure to 30–60 nm Al₂O₃ NPs mediated the scavenging activity of cells by regulating the ascorbate/glutathione pathway. Hierarchical clustering analysis indicated that ribosomal proteins were also increased upon exposure to flooding-stressed plants with 30–60 nm Al₂O₃ NPs. Mitochondrion was the target organelle of Al₂O₃ NPs under flooding-stress conditions. Mitochondrial proteomic analysis revealed that the abundance of voltage-dependent anion channel protein was increased upon exposure to flooding-stressed soybeans with 135 nm Al₂O₃ NPs, indicating the permeability of the mitochondrial membrane was increased. Furthermore, isocitrate dehydrogenase was increased upon exposure of plants to 5 nm Al₂O₃ NPs under flooding conditions. These results suggest that Al₂O₃ NPs of various sizes affect mitochondrial proteins under flooding stress by regulating membrane permeability and tricarboxylic acid cycle activity

Gel-Free/Label-Free Proteomic Analysis of Endoplasmic Reticulum Proteins in Soybean Root Tips under Flooding and Drought Stresses

Soybean is a widely cultivated crop; however, it is sensitive to flooding and drought stresses. The adverse environmental cues cause the endoplasmic reticulum (ER) stress due to accumulation of unfolded or misfolded proteins. To investigate the mechanisms in response to flooding and drought stresses, ER proteomics was performed in soybean root tips. The enzyme activity of NADH cytochrome <i>c</i> reductase was two-fold higher in the ER than other fractions, indicating that the ER was isolated with high purity. Protein abundance of ribosomal proteins was decreased under both stresses compared to control condition; however, the percentage of increased ribosomes was two-fold higher in flooding compared to drought. The ER proteins related to protein glycosylation and signaling were in response to both stresses. Compared to control condition, calnexin was decreased under both stresses; however, protein disulfide isomerase-like proteins and heat shock proteins were markedly decreased under flooding and drought conditions, respectively. Furthermore, fewer glycoproteins and higher levels of cytosolic calcium were identified under both stresses compared to control condition. These results suggest that reduced accumulation of glycoproteins in response to both stresses might be due to dysfunction of protein folding through calnexin/calreticulin cycle. Additionally, the increased cytosolic calcium levels induced by flooding and drought stresses might disturb the ER environment for proper protein folding in soybean root tips

Proteomic and Biochemical Analyses of the Cotyledon and Root of Flooding-Stressed Soybean Plants

<div><p>Background</p><p>Flooding significantly reduces the growth and grain yield of soybean plants. Proteomic and biochemical techniques were used to determine whether the function of cotyledon and root is altered in soybean under flooding stress.</p><p>Results</p><p>Two-day-old soybean plants were flooded for 2 days, after which the proteins from root and cotyledon were extracted for proteomic analysis. In response to flooding stress, the abundance of 73 and 28 proteins was significantly altered in the root and cotyledon, respectively. The accumulation of only one protein, 70 kDa heat shock protein (HSP70) (Glyma17g08020.1), increased in both organs following flooding. The ratio of protein abundance of HSP70 and biophoton emission in the cotyledon was higher than those detected in the root under flooding stress. Computed tomography and elemental analyses revealed that flooding stress decreases the number of calcium oxalate crystal the cotyledon, indicating calcium ion was elevated in the cotyledon under flooding stress.</p><p>Conclusion</p><p>These results suggest that calcium might play one role through HSP70 in the cotyledon under flooding stress.</p></div

<i>In vivo</i> biophoton emission patterns of flooding-stressed soybean plants.

<p>Two-day-old soybean plants were flooded for 2 days, after which the root and cotyledon were treated with luminal solution and photon emission was measured. Three independent biological replicate analyses of the root and cotyledon were performed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065301#pone.0065301.s004" target="_blank">Figure S4</a>). Ground photon emission by the empty Petri dish served as a blank (blue). Green lines represent biophoton emission from flooding-stressed soybean plants, while red lines represent biophoton emission from untreated soybean plants.</p

Involvement of Reactive Oxygen Species and Mitochondrial Proteins in Biophoton Emission in Roots of Soybean Plants under Flooding Stress

To understand the mechanism of biophoton emission, ROS and mitochondrial proteins were analyzed in soybean plants under flooding stress. Enzyme activity and biophoton emission were increased in the flooding stress samples when assayed in reaction mixes specific for antioxidant enzymes and reactive oxygen species; although the level of the hydroxyl radicals was increased at day 4 (2 days of flooding) compared to nonflooding at day 4, the emission of biophotons did not change. Mitochondria were isolated and purified from the roots of soybean plants grown under flooding stress by using a Percoll gradient, and proteins were analyzed by a gel-free proteomic technique. Out of the 98 mitochondrial proteins that significantly changed abundance under flooding stress, 47 increased and 51 decreased at day 4. The mitochondrial enzymes fumarase, glutathione-S-transferase, and aldehyde dehydrogenase increased at day 4 in protein abundance and enzyme activity. Enzyme activity and biophoton emission decreased at day 4 by the assay of lipoxygenase under stress. Aconitase, acyl CoA oxidase, succinate dehydrogenase, and NADH ubiquinone dehydrogenase were up-regulated at the transcription level. These results indicate that oxidation and peroxide scavenging might lead to biophoton emission and oxidative damage in the roots of soybean plants under flooding stress

Scanning electron microscopy and energy dispersive X-ray analysis.

<p>Two-day-old soybean plants were flooded for 2 days, after which the cotyledon was collected and fixed. Scanning electron micrographs and energy dispersive X-ray spectra are shown.</p

Flooding-responsive proteins identified in soybean cotyledon using LC-MS/MS.

<p>Spot no shows Spot number. Protein ID shows Phytozome protein ID. Accession no shows NCBI Accession number. MP shows matched peptides. Cov shows sequence coverage.</p

Identification of Indicator Proteins Associated with Flooding Injury in Soybean Seedlings Using Label-free Quantitative Proteomics

Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. Dissolved oxygen levels in the floodwater were decreased by the seedlings and correlated with the degree of injury. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. The analysis showed that the abundance of proteins involved in cell wall modification, such as polygalacturonase inhibitor-like and expansin-like B1-like proteins, which may be associated with the defense system, increased dependence on stress at both the protein and mRNA levels in all organs during flooding. The manner of alteration in abundance of these proteins was distinct from those of other responsive proteins. Furthermore, proteins also showing specific changes in abundance in the root tip included protein phosphatase 2A subunit-like proteins, which are possibly involved in flooding-induced root tip cell death. Additionally, decreases in abundance of cell wall synthesis-related proteins, such as cinnamyl-alcohol dehydrogenase and cellulose synthase-interactive protein-like proteins, were identified in hypocotyls of seedlings grown for 3 days after flooding, and these proteins may be associated with suppression of growth after flooding. These flooding injury-associated proteins can be defined as indicator proteins for severity of flooding stress in soybean

2-DE pattern and relative abundance of proteins in the cotyledon of flooding-stressed soybean plants.

<p>Two-day-old soybean plants were flooded for 2 days, after which proteins were extracted from the cotyledon, separated by 2-DE, and the gels were stained with CBB (A). Open circles denote protein spots showing altered staining intensity. Upward and downward arrows indicate increase or decrease in intensity, respectively. The differential abundance of proteins was determined using PDQuest software and is plotted as the relative intensity (B and C). Results are presented as the mean ± SE of relative protein intensity for gels from 3 biological replicates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065301#pone.0065301.s002" target="_blank">Figure S2</a>). Differences were compared using the Student’s <i>t</i> test (*<i>P</i><0.05, **<i>P</i><0.01). White and black bars show control and treatment, respectively. Spots numbers are the same as shown in panel A.</p

Identification of Indicator Proteins Associated with Flooding Injury in Soybean Seedlings Using Label-free Quantitative Proteomics

Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. Dissolved oxygen levels in the floodwater were decreased by the seedlings and correlated with the degree of injury. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. The analysis showed that the abundance of proteins involved in cell wall modification, such as polygalacturonase inhibitor-like and expansin-like B1-like proteins, which may be associated with the defense system, increased dependence on stress at both the protein and mRNA levels in all organs during flooding. The manner of alteration in abundance of these proteins was distinct from those of other responsive proteins. Furthermore, proteins also showing specific changes in abundance in the root tip included protein phosphatase 2A subunit-like proteins, which are possibly involved in flooding-induced root tip cell death. Additionally, decreases in abundance of cell wall synthesis-related proteins, such as cinnamyl-alcohol dehydrogenase and cellulose synthase-interactive protein-like proteins, were identified in hypocotyls of seedlings grown for 3 days after flooding, and these proteins may be associated with suppression of growth after flooding. These flooding injury-associated proteins can be defined as indicator proteins for severity of flooding stress in soybean