Cytosolic ascorbate peroxidase 2 (cAPX 2) is involved in the soybean response to flooding Fang Shi a , Ryo Yamamoto a , Satoshi Shimamura a , Susumu Hiraga a , Norikazu Nakayama a , Takuji Nakamura a , Kiyoshi Yukawa a , Mayumi Hachinohe b , Hiroshi Matsumoto b , Setsuko Komatsu a, * a National Institute of Crop Science, Kannondai 2-1-18, Tsukuba 305-8518, Japan b University of Tsukuba, Tsukuba 305-8572, Japan Received 27 May 2007; received in revised form 8 January 2008 Available online 4 March 2008 Abstract Proteomic analyses of soybean seedlings responding to flooding were conducted to identify key proteins involved. The seeds were germinated on a spongy matrix for two days, and then subjected to flooding for three days. After flooding, the total number of roots, the length of the main root, the lengths of the lateral and adventitious roots, and the fresh weight of the underground tissues of flooded soybean seedlings were significantly suppressed compared with nontreated plants. To identify the early flooding-responsive proteins, the seedling roots were used for preparing cytosolic and membrane fractions. After two-dimensional polyacrylamide gel electrophoresis and silver staining, 208 proteins were detected, and the levels of 44 were different from those of the control. The expression pattern of 10 proteins among the 44 from six different soybean cultivars confirmed that the 10 were flooding-responsive proteins. One of the 10 pro- teins was dominantly down-regulated under flooding conditions and was identified as cytosolic ascorbate peroxidase 2 (cAPX 2). North- ern-hybridization showed that the abundance of cAPX 2 transcript decreased significantly after flooding, as did the enzymatic activity of APX. These results suggest that cAPX 2 is involved in flooding stress responses in young soybean seedlings. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Soybean; Glycine max; Leguminosae; Ascorbate peroxidase 2; Flooding; Proteome; Root 1. Introduction Flooding stress is a widespread phenomenon in regions where the soil has an impermeable clay base or consists of cracking gray clay with slow drainage, where the weather is characterized by extreme rainfall, or where imperfect land planning has taken place. Most major crops exhibit no tol- erance to flooding stress, even though paddy or deepwater rice can tolerate some level of flooding. Many crops, such as canola and barley, experience serious yield losses when subjected to flooding stress (Dennis et al., 2000). The neg- ative impact of flooding on plant growth and development is a consequence of the slow diffusion rates of gases in water compared to air and the relatively low solubility of oxygen in water (Voesenek et al., 2006). Fukao and Bai- ley-Serres (2004) reported that molecular oxygen was the terminal electron acceptor in the mitochondrial electron transport chain and was required by several enzymes, including those that produce reactive oxygen species (ROS) as signaling molecules. In plants, oxygen deficiency dramatically reduces the efficiency of cellular ATP produc- tion, which has diverse ramifications for cellular metabo- lism and developmental processes. The tolerance to oxygen deprivation depends on plant cell and tissue types, 0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2008.01.007 Abbreviations: ROS, reactive oxygen species; 2D-PAGE, two-dimen- sional polyacrylamide gel electrophoresis; CBB, Coomassie brilliant blue; APX, ascorbate peroxidase. * Corresponding author. Tel.: +81 29 838 7142; fax: +81 29 838 7140. E-mail address: skomatsu@affrc.go.jp (S. Komatsu). www.elsevier.com/locate/phytochem Available online at www.sciencedirect.com Phytochemistry 69 (2008) 1295–1303 PHYTOCHEMISTRY