Abstract

To investigate the mechanism of flooding tolerance of soybean, flooding-tolerant mutants derived from gamma-ray irradiated soybean were crossed with parent cultivar Enrei for removal of other factors besides the genes related to flooding tolerance in primary generated mutant soybean. Although the growth of the wild type was significantly suppressed by flooding compared with the non-flooding condition, that of the mutant lines was better than that of the wild type even if it was treated with flooding. A two-day-old mutant line was subjected to flooding for 2 days and proteins were analyzed using a gel-free/label-free proteomic technique. Oppositely changed proteins in abundance between the wild type and mutant line under flooding stress were associated in endoplasmic reticulum according to gene-ontology categorization. Immunoblot analysis confirmed that calnexin accumulation increased in both the wild type and mutant line; however, calreticulin accumulated in only the mutant line under flooding stress. Furthermore, although glycoproteins in the wild type decreased by flooding compared with the non-flooding condition, those in the mutant line increased even if it was under flooding stress. Alcohol dehydrogenase accumulated in the wild type and mutant line; however, this enzyme activity significantly increased and mildly increased in the wild type and mutant line, respectively, under flooding stress compared with the non-flooding condition. Cell death increased and decreased in the wild type and mutant line, respectively, by flooding stress. These results suggest that the regulation of cell death through the fermentation system and glycoprotein folding might be an important factor for the acquisition of flooding tolerance in mutant soybean.

Highlights

  • IntroductionOne of the two adaptive strategies for flooding tolerance is the SNORKEL 1/2 dependent escape strategy [6]

  • To remove other factors besides the genes related to flooding tolerance in primary generated mutant soybean [16], they were crossed with parent cultivar Enrei

  • In N-linked glycoproteins, exostosin domain-containing protein significantly decreased in wild type by flooding stress; it was recovered in the mutant line even if it was under flooding stress (Tables S1–3)

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Summary

Introduction

One of the two adaptive strategies for flooding tolerance is the SNORKEL 1/2 dependent escape strategy [6] It promotes the internode elongation through the stimulation of gibberellin biosynthesis in deep-water rice under flooding stress, and thereby enabling rice grows upward to the water surface for air exchange [6]. Another adaptive strategy is the SUBMERGENCE1 dependent quiescence strategy [7]. Using the primary generated flooding-tolerant mutant, gel-based proteomic analysis was performed, indicating that activation of the fermentation system in the early stages of flooding is an important factor for the acquisition of flooding tolerance in soybean [16]. Proteomic results were subsequently confirmed by immunoblot, enzyme activity, and physiological analyses

Morphological Analysis of Mutant Soybean under Flooding Stress
Experimental
Evans-Blue Staining with Mutant Soybean under Flooding Stress
3.3.Discussion
Glycoprotein Folding Is Related to the Mechanism of Flooding Tolerance
Cell Death in Soybean Root Is Related to Flooding Tolerance
Plant Material and Experimental Design
Protein Identification Using Nano-Liquid Chromatography Mass Spectrometry
Mass Spectrometry Data Analysis
Differential Analysis of Proteins Using Mass Spectrometry Data
Immunoblot Analysis
ADH Activity Assay
Conclusions
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