Proteomic analyses of soybean seedlings responding to flooding were conducted to identify proteins involved in such response. Soybean was germinated for 48 h and then subjected to flooding stress for 6-48 h. Proteomic analysis of hypocotyl and root was used in a time-dependent manner, and altered proteins were identified using soybean protein data file constructed for this research. Under flooding stress, 35 proteins were up-regulated, whereas 16 proteins were down-regulated at a 24-h time point. Changes in energy generation was recognized because several glycolytic enzymes were up-regulated. General stress response was also shown to occur as various reactive oxygen species scavengers were up-regulated. Other identified proteins with diverse functional categories suggest that flooding stress includes not only hypoxic stress, but also other stresses such as weak light, disease, and water stresses. In addition, proteins with unknown functions were shown to be positioned as hubs which activate other proteins in system response networks by protein-protein interaction analysis, suggesting that this type of interaction analysis is useful for screening of important factors in plant response to environmental stresses.

Alcohol dehydrogenase (Adh) is the key enzyme in alcohol fermentation. We analyzed Adh expression in order to clarify the role of Adh of soybeans (Glycine max) to flooding stress. Proteome analysis confirmed that expression of Adh is significantly upregulated in 4-day-old soybean seedlings subjected to 2 days of flooding. Southern hybridization analysis and soybean genome database search revealed that soybean has at least 6 Adh genes. The GmAdh2 gene that responded to flooding was isolated from soybean cultivar Enrei. Adh2 expression was markedly increased 6 h after flooding and decreased 24 h after floodwater drainage. In situ hybridization and Western blot indicated that flooding strongly induces Adh2 expression in RNA and protein levels in the root apical meristem. Osmotic, cold, or drought stress did not induce expression of Adh2. These results indicate that Adh2 is a flooding-response specific soybean gene expressed in root tissue.

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Abstracte soybean is relatively sensitive to disturbances arising from ooding before its emergence from the soil. When young soybean seedlings at an early stage are transferred to ooding anaerobic conditions, alcohol dehydrogenase (Adh) mRNA and Adh protein increase temporarily in the root tips, where active cell division demands high energy production. Since there is little information on the signi cance of the up-regulation of Adh for the tolerance of soybeans to ooding stress, we examined the response to ooding in transgenic soybean lines in which the soybean Adh (GmAdh2) gene was introduced under the control of a constitutive promoter. Acquired transgenic soybean seeds from one out of 14 transgenic lines were subjected to ooding stress. Growth inhibition of soybean seedlings caused by ooding stress was reduced in soybeans with the GmAdh2 transgene. Protein analysis and enzyme assay at the early stage of growth of the soybean seedlings con rmed that Adh expressions and activities in transgenic soybeans were increased compared to control soybeans. ese results indicated that the introduced GmAdh2 gene might have induced some change in glycolysis and alcohol fermentation, and improved the germination of transgenic soybeans under ooding stress. Key words:Alcohol dehydrogenase, ooding, soybean, transformation, transgenic soybean.Soybeans are generally intolerant of flooding stress. In many regions of Japan, soybean seeds are sown in a paddy eld during the summer-rainy season, and excess rainfall after sowing can often lead to soil flooding. Flooding a er sowing causes severely decreased crop yields. ese lower yields may result from the collapse of cotyledons due to rapid imbibitions of water (Nakayama et al. 2004) and from serious damage to the root system. Accordingly, it is important to understand the mechanism of the ooding stress responses in order to improve crop yields. However, the flooding stress responses in soybeans are not well characterized.Studies of the responses of soybean seedlings to flooding stress showed that flooding inhibits root elongation and hypocotyl pigmentation (Hashiguchi et al. 2009) and a ects the expression of some proteins involved in the processes of fermentation (Russell et al. 1990), the scavenging of reactive oxygen species (Shi et al. 2008), glycolysis, protein storage, and defense against disease (Hashiguchi et al. 2009). Since alcoholic fermentation is the major fermentation pathway of glycolysis in anaerobic plants (Rees et al. 1987;Komatsu et al. 2010a), the mechanism of tolerance to ooding stress should include the upregulation of genes engaged in glycolysis and alcohol fermentation. Under low oxygen stress conditions, plants activate alcohol fermentation in which pyruvate is used as a starting substrate. Pyruvate decarboxylase (Pdc, EC 4.1.1.1) catalyzes the rst step and converts pyruvate to acetaldehyde.en, alcohol dehydrogenase (Adh, EC 1.1.1.1) converts acetaldehyde to ethanol, with the concomitant regeneration of NAD + for glycolysis. Adh is a fermentative enzyme that...