Differential gene expression of two alternative oxidase genes under dehydration stress in common wheat

Nobuyuki Mizuno and Shigeo Takumi

Laboratory of Plant Genetics, Graduate School of Agricultural Science, Kobe University, Nada-ku, Kobe 657-8501, Japan

Corresponding author: Shigeo Takumi

E-mail: takumi@kobe-u.ac.jp

Reactive oxygen species (ROS) are generated by various stress and target to mitochondria under the low temperature and drought conditions (Bartoli et al. 2004). Plant mitochondria possess a unique respiratory pathway, the cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive alternative pathway, besides the main cytochrome pathway (Henry and Nyns 1975). The mitochondrial alternative pathway is a non-phosphorylating electron transport pathway branching from the cytochrome pathway at the ubiquinone pool, and the electron flow through the pathway reduces oxygen to water (Siedow 1982). Alternative oxidase (AOX) is a terminal oxidase functioning as a key enzyme in the mitochondrial alternative pathway (Vanlerberghe and McIntosh 1997). Many conditions inducing oxidative stress increase the AOX activity, suggesting that AOX can function as an antioxidant enzyme and act to alleviate the ROS accumulation which occurs due to impaired or restricted respiration activity in mitochondria (Moore et al. 2002; Sugie et al. 2006).

Two non-homoeologous AOX genes, WAOX1a and WAOX1c, were previously isolated from common wheat (Takumi et al. 2002). Our previous studies revealed that the two AOX genes were transcriptionally activated by low temperature treatment and functioned to alleviate ROS accumulation under the low temperature conditions (Sugie et al. 2006; Mizuno et al. 2008). The wheat AOX genes are valuable to monitor the ROS generation in wheat cells towards necrotic cell death (Sugie et al. 2007; Mizuno et al. 2010). It was recently reported that wheat AOX protein levels increased under drought stress (Bartoli et al. 2005). To compare the expression patterns of those two AOX genes under dehydration stress, reverse transcriptase (RT)-PCR analysis was conducted in this study.

Two common wheat (Triticum aestivum L.) cultivars, Chinese Spring (CS) and Mironovskaya 808 (M808), were used in this study. No significant difference of dehydration tolerance was observed between CS and M808 (Egawa et al. 2006). For H₂O₂ detection, 3-3’-diaminobenzidine tetrahydrochloride (DAB) (Wako, Osaka, Japan) was used, and leaf samples were infiltrated in 2% mg ml^-1 DAB solution (pH3.8) for 8 hours. The infiltrated leaves were then treated with 100% ethanol for chlorophyll removal. For gene expression study, RT-PCR analysis was conducted. Total RNA isolation and the first strand cDNA synthesis were performed referred to Egawa et al. (2006). The gene-specific primer sets are listed on Table 1. Advantage 2 polymerase mix (BD Biosciences, San Jose, CA, USA) was used only for the WAOX1a and WAOX1c amplification. The PCR condition for WAOX1a and WAOX1c was demonstrated according to Sugie et al. (2007), and 30 cycles of PCR were performed in the exponential range of amplification. The wheat ubiquitin gene was used as an internal control. The PCR-amplified products were fractionated by electrophoresis through a 1.5% agarose gel and stained with ethidium bromide.

DAB infiltration assay showed that dehydrated leaves more abundantly accumulated H₂O₂ than the seedling leaves of CS and M808 under the normal condition (Fig. 1). The ROS generation was observed at least after six hours of dehydration treatment in wheat seedling leaves. Therefore, dehydration stress rapidly induced the H₂O₂ accumulation in wheat cells.

To compare the expression patterns of the two AOX genes under dehydration stress, RT-PCR was performed in CS and M808 using their gene specific primers. Transcript accumulation levels of WAOX1a gradually increased during dehydration in CS (Fig. 2). However, the WAOX1c transcript level was once enhanced by dehydration stress, and then repressed after six hours of dehydration stress in CS. Transcript accumulation levels of WHLP and TaMRP11, which were other nuclear genes targeting to mitochondria (Mizumoto et al. 2004; Handa et al. 2001; Sugie et al. 2007), gradually decreased after six hours of dehydration stress in CS. These expression patterns of the four examined genes were also confirmed in the dehydration stress-treated M808 seedlings (data not shown).

The two wheat AOX genes were up-regulated by dehydration stress, accompanied with H₂O₂ generation (Fig. 1; Fig. 2). This observation suggested that wheat AOX acts for the ROS alleviation under dehydration stress as well as low temperature stress (Sugie et al. 2006; Mizuno et al. 2008). High accumulation levels of the WAOX1a transcripts might infer the significant function of WAOX1a to alleviate the ROS accumulation during dehydration stress. Decrease in the WAOX1c, WHLP and TaMRPL11 transcript levels under the dehydration stress condition indicated that the mitochondrial function might be impaired by dehydration stress. It was previously reported that the two wheat AOX genes showed differential expression patterns not only under low temperature stress and but also in the necrosis-exhibiting wheat hybrids (Sugie et al. 2007; Mizuno et al. 2008; Mizuno et al. 2010). An increased level of the WAOX1a transcripts was observed in the necrosis-exhibiting wheat hybrids, whereas there was no significant change in the WAOX1c transcript accumulation levels (Sugie et al. 2007). The WAOX1a transcript showed gradual accumulation in the low temperature-treated seedling leaves of wheat cultivars, and the low temperature-responsiveness of WAOX1c was less than that of WAOX1a (Mizuno et al. 2008). Together with these results, it was concluded that the two AOX genes, WAOX1a and WAOX1c, are differentially regulated at the gene expression level under various abiotic stress with ROS generation, and that WAOX1a mainly acts to alleviate the ROS accumulation in mitochondria.

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