Production of reactive oxygen species under low temperature condition in seedling leaves of common wheat

 

Nobuyuki Mizuno, Atsushi Sugie 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

 

 

Abstract

To study physiological processes of development of freezing tolerance in common wheat, we compared production of reactive oxygen species (ROS) under low temperature using two cultivars differing in freezing tolerance. The ROS production was observed at mitochondria. During cold acclimation, a freezing-tolerant cultivar produced the smaller amount of ROS than a freezing-sensitive cultivar. This result suggested that repression of ROS production might contribute on the development of freezing tolerance in wheat.

 

 

The electron transport chains of mitochondria and chloroplasts are important sources of reactive oxygen species (ROS) in plant cells, and at least some of the effects of low temperature stress are mediated by ROS (Bowler et al. 1992). To keep ROS at low steady-state levels, antioxidant enzymes function under the low temperature stress conditions (Prasad et al. 1994a; Prasad 1996). Competence and stability of mitochondria are important for plants to survive low temperature stress, because mitochondria are the source and target of oxidative stress (Prasad et al. 1994b). 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 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 alternative pathway (Vanlerberghe and McIntosh 1997). Many conditions causing oxidative stress induce the AOX activity, suggesting that AOX can function as a mechanism to decrease the formation of ROS produced as a result of impaired or restricted respiration activity (Moore et al. 2002; Sugie et al. 2006). A variety of biotic and abiotic stresses have been shown to give a negative impact on the cytochrome pathway and induce AOX (Purvis and Shewfelt 1993; Vanlerberghe et al. 2002).

 

Cold acclimation is an adaptive process for acquiring cold/freezing tolerance in wheat. To compare the ROS amount during cold acclimation in two wheat cultivars showing distinct levels of freezing tolerance, a winter cultivar ‘Mironovskaya 808’ (abbreviated as M808) and a spring cultivar ‘Chinese Spring’ (CS) of common wheat (Triticum aestivum L.) were used as a cold/freezing tolerant and susceptible accessions, respectively (Ohno et al. 2001). M808 was bred in Mironovskaya Institute, Ukraine, and reported to be the hardiest winter cultivar among tetraploid and hexaploid wheats tested for freezing tolerance (Veisz and Sutka 1990). Our previous studies also showed the much higher freezing tolerance of M808 compared with that of CS by the simple one-point assay (Ohno et al. 2001; Kume et al. 2005). Seeds of M808 and CS were planted in pots (25 cm x 12 cm in width and 12 cm in depth) with soil, and were grown in a growth cabinet at 22˚C with a 16 h photoperiod (the standard condition). For low temperature treatment, 2-week-old seedlings of CS and M808 grown under the standard condition were transferred to 4˚C.

 

For detection of ROS, two types of fluorescent dye were used according to Sakamoto et al. (2005). Intracellular ROS, especially H2O2, in the seedling leaves were measured using 2,7-dichlorofluorescein diacetate (DCF) (Molecular Probe, USA). Leaf segments were incubated for 30 min in the presence of 5 µM DCF. DCF fluorescence images were excited at 488 nm and detected using NIBA filter (515-550 nm). A Nikon TE300 inverted microscope was used for fluorescence microscopy. The images were captured by an intensified-CCD camera (MicroMAX; Princeton Instruments, Trenton, NJ) and fluorescence intensity was quantified with Metamorph software (Universal Imaging Corp., West Chester, PA) after subtraction of background fluorescence (measured in the absence of fluorescent probes) in each image. Mitochondria-specific ROS was measured using dihydrorhodamine 123 (DHR) (Molecular Probe, USA). For the DHR staining, leaf segments were incubated for 15 min in the presence of 1 µM DHR solution. The residual cytosolic fraction of the dye was essentially eliminated when the leaf segments were kept in distilled water for an additional 3-4 hr after incubation. Fluorescence images of DHR were detected using NIBA filter (515-550 nm). For the H2O2 detection, 3-3’-diaminobenzidine tetrahydrochloride (DAB) (Wako, Osaka, Japan) was additionally used for detection of ROS. Leaf samples were infiltrated in 2% mg ml-1 DAB solution (pH3.8) for 8 hr, and then treated with 100% ethanol for chlorophyll removal.

 

Comparison of epidermal cell layers of DCF-stained seedling leaves showed that the number of H2O2-produced cells in the low temperature-treated M808 was much larger than that in non-treated control (Fig. 1A and 1B), indicating massive, intracellular ROS-production via low temperature treatment. To examine intracellular localization of ROS, DHR staining was performed. The DHR fluorescence clearly indicated that mitochondria were at least one of organelles producing ROS under low temperature (Fig. 1C). The ROS production in wheat mitochondria was also previously reported under the drought stress condition (Bartoli et al. 2004). DAB infiltration also showed that low temperature-treated seedlings more abundantly accumulated H2O2 than non-treated control in both CS and M808 (Fig. 1D). To confirm the cultivar difference of the ROS production, DCF-fluorescence was quantified and the average levels of the DCF-fluorescence per leaf were compared between CS and M808 leaves (Fig. 1E). Low temperature treatment slightly increased the ROS production in both cultivars. One-day low temperature treatment induced 1.20- and 1.09-times increase of the ROS production in CS and M808 compared with the normal temperature (22˚C), respectively. ROS was slightly produced at the higher level under the low temperature condition (4˚C) in CS than in M808, although the difference was not significant.

 

Cultivar difference of freezing tolerance could be at least partly caused by the differential accumulation levels of Cor/Lea transcripts during cold acclimation (Vágújfalvi et al. 2000; Kobayashi et al. 2004). During the cold acclimation, M808 produced the smaller amount of ROS than CS (Fig. 1), which suggesting the other factor affecting the cultivar difference of cold/freezing tolerance in wheat. Our recent study showed that the respiration capacity of alternative pathway significantly increased in M808 under the low temperature conditions, and that the higher levels of the alternative pathway capacity in M808 seemed to be coupled with the abundant accumulation of the AOX transcripts during cold acclimation (Mizuno et al. 2007).

 

Together with these observations, it should be suggested that the high levels of the AOX transcript accumulation and the alternative pathway activity efficiently suppressed the ROS formation during cold acclimation and contributed on development of the cold/freezing tolerance in M808. Actually, AOX can function to prevent the ROS formation from an over-reduced ubiquinone pool in plant mitochondria (Purvis and Shewfelt 1993), and expression levels of the AOX gene are corresponding to suppression of the ROS production in transgenic tobacco cells (Umbach et al. 2005). Two non-homoeologous genes, Waox1a and Waox1c, encoding the AOX proteins were previously isolated from common wheat and were commonly responsive to low temperature (Takumi et al. 2002). Our recent study also showed that overexpression of the Waox1a gene alleviates oxidative stress when the cytochrome pathway of respiration is inhibited under low temperature stress conditions in transgenic Arabidopsis (Sugie et al. 2006). The altered levels of Arabidopsis AOX protein resulted in altered leaf growth phenotypes under low temperature condition in transgenic plants (Fiorani et al. 2005). These recent studies suggest that repression of ROS production during cold acclimation might contribute at least partly on the development of freezing tolerance in wheat, and that the alternative pathway might be associated with the ROS repression.

 

 

Acknowledgements

We thank Drs. S. Mayama and M. Sakamoto for their technical support of the ROS detection. We are also grateful to Dr. C. Nakamura for use of his facilities. The work was supported by a ‘grant-in-aid’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to ST, no. 17780005).

 

 

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