Defense/stress responses elicited in rice seedlings exposed to the gaseous air pollutant sulfur dioxide

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Abstract

This report examines for the first time, the response of rice (Oryza sativa japonica-type cv. Nipponbare) seedlings exposed to the air pollutant sulfur dioxide (SO2). Distinctive reddish-brown necrotic spots and interveinal browning appeared on the leaf surface after exposure to SO2, over control, partly reminiscent of the hypersensitive reaction lesions. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting analysis revealed strong induction of ascorbate peroxidase(s), and changes in cysteine proteinase inhibitors (‘phytocystatins’)-like proteins. Employing classical two-dimensional electrophoresis (2-DE) followed by amino acid sequencing, we identified several changes in the 2-DE protein profiles of SO2-fumigated leaves. Most prominent changes in leaves were the induced accumulation of a pathogenesis-related (PR) class 5 (OsPR5) protein, three PR 10 class proteins (OsPR10s), a novel ATP-dependent CLP protease, and an unknown protein. Subsequent Northern analysis showed potent accumulation of OsPR5 and OsPR10 transcripts in leaves. Finally, mass spectrometry analysis revealed a strong production of phytoalexins, sakuranetin and momilactone A in SO2 stressed leaves. Our results not only demonstrate the highly damaging effect of SO2, but also identify SO2 triggered multiple events linked with defense/stress response in the leaves of rice seedlings.

Introduction

Environmental pollution by sulfur-containing compounds, e.g. sulfur dioxide (SO2), hydrogen sulfide, sulfite (SO32−), and sulfate ions (SO42−), is a serious problem for the global environment. The gaseous SO2 can occur naturally as a result of volcanic activity, the decay of dead animals and plants and the effect of lightning. However, in the air, SO2 mainly comes from activities such as the burning of coal and oil at power plants or from ore smelting, influencing human health and the global ecological system of animal and plant life (Wellburn, 1994; Murray, 1997; Noji et al., 2001). There is growing evidence that the initiation of a free radical generating mechanism by SO2 results in peroxidative destruction of cellular constituents (Shimazaki et al., 1980; Norman et al., 2001). The multiple effects of both SO2 and SO32− on the plant include pigment destruction, depletion of cellular lipids and peroxidation of polyunsaturated fatty acids (Khan and Malhotra, 1977; Sandmann and Gonzales, 1989 and references therein); therefore the toxicity of SO2 is thought to result from generation of reactive oxygen species (ROS).

Although many monocotyledonous species, including grasses and the cereal crops such as barley, wheat, oats and maize have been the subject of detailed investigations in their response(s) to SO2 (Spedding, 1969; Mudd, 1975; Ashenden and Mansfield, 1978; Ayazloo et al., 1982; Bell, 1982; Gould et al., 1988; Navari-Izzo et al., 1989; Price and Long, 1989; Murray and Wilson, 1990; Deepak and Agrawal, 1999; Kondo, 2002, and references therein), the rice (Oryza sativa) plant, an enormously important monocot research model whose draft genome sequence has recently been released (Goff et al., 2002), has received less attention. In this study, we tested the hypothesis that two-week-old rice seedlings would respond to SO2 fumigation by inducing various biochemical/molecular changes associated with the defense/stress response, including those involving mechanisms affecting the inactivation of ROS. As proteomics is a powerful tool in understanding which proteins are present in particular tissue under given condition (Porubleva and Chitnis, 2000), we employed immunoblotting, classical 2-DE and amino acid sequencing (Rakwal et al., 1999; Hajduch et al., 2001) to monitor the changes in protein profiles. In addition, Northern blot analysis and high-pressure liquid chromatography mass spectrometry (HPLC-MS) was used to identify changes in mRNA expression profiles of some defense/stress-related genes and induced accumulation of secondary metabolites, in order to assess the deleterious effect of SO2 in rice seedlings.

Section snippets

Plant material and fumigation with SO2

Rice (Oryza sativa L. cv. Nipponbare) seedlings were grown for 2 weeks under white fluorescent light (wavelength 390–500 nm, 150 μmol m−2 s−1, 12 h photoperiod) at 25 °C and 70% relative humidity (RH) as previously reported (Agrawal et al., 2001; Rakwal et al., 2001a, Rakwal et al., 2001b, Rakwal et al., 2001c). At this stage, the seedling is composed of 4 leaves, a shoot (leaf sheath) and roots. After completing the 12 h dark cycle, pots containing the seedlings (50 seedlings/pot) were

SO2 induced damage to rice seedling leaves

The first visual symptoms of SO2 induced damage to the rice seedlings were observed in the leaves, where slight discolored light brownish spots were found to appear on the upper side of the leaves around 60 h after exposure to SO2, and at 72 h post-treatment, intensely reddish-brown colored ‘necrotic lesions’, and interveinal browning were apparent almost all over the leaf surface with SO2 (Fig. 1, 3rd and 4th leaf, 72 h SO2; CON represents the control for the same time-period). On the other

Discussion

The present study was carried out to identify how SO2 affects rice seedlings, with an immediate goal to look at the symptomology, changes in proteins, genes and secondary metabolites related to rice self-defense response(s). Induction of lesions upon exposure to SO2 is reminiscent of the hypersensitive reaction (HR), which is believed to be apart of the programmed cell death (PCD) during pathogen attack, and involves generation of the ROS. It is believed that SO2 might involve two simultaneous

Acknowledgements

R.R. is a Japan Society for Promotion of Science (JSPS) fellow working at AIST.

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