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

Oh M, Nanjo Y, Komatsu S - Front Plant Sci (2014)

Bottom Line: Soybean is sensitive to flooding stress and exhibits reduced growth under flooding conditions.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.Fermentation and glycolysis-related proteins were increased in response to flooding; however, these proteins were not affected by calcium supplementation.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences and Bioengineering, Graduate School of Life and Environmental Sciences, University of Tsukuba Tsukuba, Japan ; National Institute of Crop Science, National Agriculture and Food Research Organization Tsukuba, Japan.

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.

No MeSH data available.


Evaluation of cell death in flooding-stressed soybean root tips treated with calcium. Two-day-old soybeans were flooded without (dark blue) or with 1 (red), 5 (light green), 10 (purple), and 50 mM CaCl2 (light blue) for 2, 4, and 6 days. The roots were stained with 0.25% Evans blue dye, which was then extracted and measured spectroscopically at 600 nm. (A) Photographs show soybeans after 2, 4, and 6 days of flooding. Bars indicate 10 mm. (B) Absorbance of Evans blue in root tips at the indicated time points. Data are means ± SE from three independent biological replications. Means with the same letter are not significantly different according to ANOVA (P < 0.05).
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Figure 2: Evaluation of cell death in flooding-stressed soybean root tips treated with calcium. Two-day-old soybeans were flooded without (dark blue) or with 1 (red), 5 (light green), 10 (purple), and 50 mM CaCl2 (light blue) for 2, 4, and 6 days. The roots were stained with 0.25% Evans blue dye, which was then extracted and measured spectroscopically at 600 nm. (A) Photographs show soybeans after 2, 4, and 6 days of flooding. Bars indicate 10 mm. (B) Absorbance of Evans blue in root tips at the indicated time points. Data are means ± SE from three independent biological replications. Means with the same letter are not significantly different according to ANOVA (P < 0.05).

Mentions: To investigate the effects of calcium on morphological changes induced by flooding in soybean and evaluate the corresponding degree of root tip damage, 2-day-old soybeans were flooded for 2, 4, and 6 days without or with 1, 5, 10, and 50 mM CaCl2. The total length of root including hypocotyl under flooding with various concentrations of CaCl2 was clearly longer than that under flooding without CaCl2 (Figure 1A). Although no marked differences in root length were detected between flooded soybean in the presence or absence of 1 mM CaCl2, the length after treatment with 10 and 50 mM CaCl2 was significantly longer than that under flooding without CaCl2 (Figure 1B). Consistent with this finding, the root weight of flooding-treated soybeans was also higher for plants exposed to CaCl2 during the treatment period (Figure 1B). The length and weight of root including hypocotyl were measured after each treatment in biological triplicates to assess reproducibility (Supplemental Figures 2A–C). The length and weight of root were gradually increased in 2-, 4-, and 6-day flooded soybean with 10 and 50 mM CaCl2. In particularly, 2-day-flooded soybean was most affected by CaCl2 (Figure 1). The flooding-treated soybeans were also stained with Evans blue dye to evaluate cell death (Figure 2). The degree of staining in root tips was dependent on the CaCl2 concentration and treatment period (Figure 2A). Cell death in the root tip was severely induced by flooding without CaCl2 compared to that in the presence of 50 mM CaCl2 (Figure 2B). The Evans blue staining was also performed in biological triplicate experiments to assess reproducibility (Supplemental Figures 3A–C). Even though cell death in the root tip was significantly suppressed by flooding with CaCl2 in 4 and 6 days, the degree of staining in root tips was most affected by CaCl2 in 2-day-flooded soybean (Figure 2).


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

Oh M, Nanjo Y, Komatsu S - Front Plant Sci (2014)

Evaluation of cell death in flooding-stressed soybean root tips treated with calcium. Two-day-old soybeans were flooded without (dark blue) or with 1 (red), 5 (light green), 10 (purple), and 50 mM CaCl2 (light blue) for 2, 4, and 6 days. The roots were stained with 0.25% Evans blue dye, which was then extracted and measured spectroscopically at 600 nm. (A) Photographs show soybeans after 2, 4, and 6 days of flooding. Bars indicate 10 mm. (B) Absorbance of Evans blue in root tips at the indicated time points. Data are means ± SE from three independent biological replications. Means with the same letter are not significantly different according to ANOVA (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4202786&req=5

Figure 2: Evaluation of cell death in flooding-stressed soybean root tips treated with calcium. Two-day-old soybeans were flooded without (dark blue) or with 1 (red), 5 (light green), 10 (purple), and 50 mM CaCl2 (light blue) for 2, 4, and 6 days. The roots were stained with 0.25% Evans blue dye, which was then extracted and measured spectroscopically at 600 nm. (A) Photographs show soybeans after 2, 4, and 6 days of flooding. Bars indicate 10 mm. (B) Absorbance of Evans blue in root tips at the indicated time points. Data are means ± SE from three independent biological replications. Means with the same letter are not significantly different according to ANOVA (P < 0.05).
Mentions: To investigate the effects of calcium on morphological changes induced by flooding in soybean and evaluate the corresponding degree of root tip damage, 2-day-old soybeans were flooded for 2, 4, and 6 days without or with 1, 5, 10, and 50 mM CaCl2. The total length of root including hypocotyl under flooding with various concentrations of CaCl2 was clearly longer than that under flooding without CaCl2 (Figure 1A). Although no marked differences in root length were detected between flooded soybean in the presence or absence of 1 mM CaCl2, the length after treatment with 10 and 50 mM CaCl2 was significantly longer than that under flooding without CaCl2 (Figure 1B). Consistent with this finding, the root weight of flooding-treated soybeans was also higher for plants exposed to CaCl2 during the treatment period (Figure 1B). The length and weight of root including hypocotyl were measured after each treatment in biological triplicates to assess reproducibility (Supplemental Figures 2A–C). The length and weight of root were gradually increased in 2-, 4-, and 6-day flooded soybean with 10 and 50 mM CaCl2. In particularly, 2-day-flooded soybean was most affected by CaCl2 (Figure 1). The flooding-treated soybeans were also stained with Evans blue dye to evaluate cell death (Figure 2). The degree of staining in root tips was dependent on the CaCl2 concentration and treatment period (Figure 2A). Cell death in the root tip was severely induced by flooding without CaCl2 compared to that in the presence of 50 mM CaCl2 (Figure 2B). The Evans blue staining was also performed in biological triplicate experiments to assess reproducibility (Supplemental Figures 3A–C). Even though cell death in the root tip was significantly suppressed by flooding with CaCl2 in 4 and 6 days, the degree of staining in root tips was most affected by CaCl2 in 2-day-flooded soybean (Figure 2).

Bottom Line: Soybean is sensitive to flooding stress and exhibits reduced growth under flooding conditions.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.Fermentation and glycolysis-related proteins were increased in response to flooding; however, these proteins were not affected by calcium supplementation.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences and Bioengineering, Graduate School of Life and Environmental Sciences, University of Tsukuba Tsukuba, Japan ; National Institute of Crop Science, National Agriculture and Food Research Organization Tsukuba, Japan.

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.

No MeSH data available.