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Plant Molecular Biology

, Volume 77, Issue 3, pp 309–322 | Cite as

Characterization of a novel flooding stress-responsive alcohol dehydrogenase expressed in soybean roots

  • Setsuko Komatsu
  • Deschamps Thibaut
  • Susumu Hiraga
  • Mikio Kato
  • Mitsuru Chiba
  • Akiko Hashiguchi
  • Makoto Tougou
  • Satoshi Shimamura
  • Hiroshi Yasue
Article

Abstract

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.

Keywords

Alcohol dehydrogenase Gene expression Flooding Hypoxic stress Soybean 

Abbreviations

Adh

Alcohol dehydrogenase

2-DE

Two-dimensional polyacrylamide gel electrophoresis

MS

Mass spectrometry

IEF

Isoelectric focusing

CBB

Coomassie brilliant blue

Notes

Acknowledgments

This work was supported by the grants from National Agriculture and Food Research Organization, Japan. The authors appreciate Dr. K. Yukawa, Dr. Y. Nanjo, Dr. K. Nishizawa, Dr. T. Nakamura and Mr. H Saito for their experimental support and helpful discussions for this research.

Supplementary material

11103_2011_9812_MOESM1_ESM.pdf (275 kb)
Suppl. Figure 1. Mascot Search Results for MS analysis of protein spots induced by flooding (PDF 274 kb)
11103_2011_9812_MOESM2_ESM.jpg (23 kb)
Suppl. Figure 2 (JPG 23 kb)
11103_2011_9812_MOESM3_ESM.doc (90 kb)
Suppl. Figure 3. Amino acid sequence alignment of Adh and Adh-like proteins. (A) Alignment of soybean Adh-2 (Glyma04g41990.1) and horse liver Adh (EcLADH). (B) Alignment of Adh proteins from soybean, Arabidopsis, rice and maize. (C) Alignment of 27 soybean Adh and Adh-like proteins. Identical and similar amino acids are represented as “*” and “:” or “.”, respectively. In (A), the circles above the sequence denote amino acid residues associated with the active site as revealed by studies of horse liver Adh (Hammes-Schiffer and Benkovic 2006). Cysteine ligands in the structural zinc site are indicated by a triangle. Corresponding residues of plant Adh and Adh-like proteins were similarly marked in (B) and (C) (DOC 90 kb)
11103_2011_9812_MOESM4_ESM.pdf (125 kb)
Suppl. Figure 4. Nucleotide sequence alignment of soybean Adh genes that are closely related to Adh2. (A) Alignment of genomic regions corresponding to the probe. (B) Alignment of cDNA sequences. All sequences analyzed were retrieved from the public genome sequence database for the cv. Williams82 (http://www.phytozome.net/soybean). Shaded nucleotides denote those the most conserved among the soybean Adh genes indicated. The sequence of the partial protein coding region, Glyma14g24860.1 cDNA, which is available to the public, was analyzed (PDF 124 kb)
11103_2011_9812_MOESM5_ESM.pdf (97 kb)
Suppl. Figure 5. The pattern of agarose gel electrophoresis of PCR products. Prior to realtime RT-PCR analysis, we selected primers from 3’ untranslated regions except for Adh-2 whose 3’ UTR primers amplified multiple bands for unknown reason. Specificity of selected primers was verified by checking melt curves of amplified fragments and agarose gel electrophoresis of PCR products (PDF 96 kb)
11103_2011_9812_MOESM6_ESM.pdf (15 kb)
Suppl. Figure 6. Comparison of Adh expression in soybeans and rice. Adh expression determined by quantitative real-time RT-PCR analysis. Two-day-old soybean and rice seedlings were flooded for 1 day and then the floodwater was drained. Soybean hypocotyls and roots, and rice roots were collected after 1 day of flooding or 1 day after floodwater removal. Adh expression was determined by quantitative real-time RT-PCR analysis. White and black rectangles show control and treated samples, respectively. Values shown are the means of mRNA values obtained from three replicates, and the standard error is denoted by error bars (PDF 15 kb)
11103_2011_9812_MOESM7_ESM.pdf (35 kb)
Supplementary material 7 (PDF 35 kb)

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Setsuko Komatsu
    • 1
  • Deschamps Thibaut
    • 1
    • 2
  • Susumu Hiraga
    • 1
  • Mikio Kato
    • 3
  • Mitsuru Chiba
    • 4
    • 5
  • Akiko Hashiguchi
    • 1
  • Makoto Tougou
    • 1
  • Satoshi Shimamura
    • 1
  • Hiroshi Yasue
    • 4
  1. 1.National Institute of Crop ScienceTsukubaJapan
  2. 2.University Pierre et Marie Curie UniversityParisFrance
  3. 3.Osaka Prefecture UniversityOsakaJapan
  4. 4.National Institute of Agrobiological SciencesTsukubaJapan
  5. 5.University of TsukubaTsukubaJapan

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