The effect of ozone on rice seedlings
under normal conditions, and then some plants were transferred to an ozone chamber. The ozone concentration was set to 0.2 ppm, a level that is sometimes exceeded during summer months in the region around Tokyo, says Rakwal. To examine early stages of the plant’s response, the team took samples at 1, 12, and 24 hours of exposure. Gene expression was monitored at all three time points with microarrays, but proteomics and metabolomics
Ground-level ozone is a pollutant that can cause lung irritation, chest pain, and other symptoms in humans. To escape some of the effects of ozone, we can walk indoors, but what about plants, our distant—and stationary— cousins? Scientists know that ozone damages plants, such as rice, and reduces their yields. Because rice is one of the most important food crops in Asia, Randeep Rakwal at the National Institute of Advanced Industrial Science and Technology (AIST; 0.2 ppm Japan) and the Research Labora03 tory for Agricultural Biotechnology and Biochemistry (RLABB; Nepal), was surprised to discover that no one had studied how ozone affects the plant on a molecular level. So, he and colleagues at AIST; RLABB; Sejong University (South Korea); Akita Prefectural University; the National Institute of Agrobiological Sciences; the National Institute for Environmental Studies (all in Japan); and the National Institute of Occupational Safety and Health, Japan, took a systems biology approach to address the topic. In JPR DNA microarray 2DE and MS (2008, 7, 2980–2998), the scientists CE/MS analysis report that various genes, proteins, and metabolites are differentially regulated in rice seedlings exposed Integrative interpretation to a high concentration of ozone of ozone effect compared with controls. Some studies on the plant ozone Let’s get together. Transcriptomics, proteomics, and response have been conducted metabolomics data were integrated to figure out the with Arabidopsis thaliana, but few molecular effects of ozone on rice. have been conducted with rice. Rakwal says that some researchers think that if a question is addressed analyses were performed only with the in A. thaliana, a model organism that samples taken at 24 hours. This strategy belongs to the dicot classification of was implemented to get the most bang flowering plants, then there is no need for the researchers’ budget; they reato pursue it in other plants, such as rice, soned that the effects on proteins and which is a monocot. (Cotton, sunflower, metabolites would occur later than the soybeans, and other broad-leafed gene expression changes. plants fall into the dicot class, whereas Among the 1535 genes that were difwheat, maize, and other grasses are ferentially regulated in ozone-treated monocots.) However, he explains that rice compared with controls, the monocot and dicot structures are difresearchers found genes encoding for ferent, “and when we look at the genes, several transcription factors, signaling now we are realizing that a particular proteins, and metabolic factors. Many gene might have a particular regulaof the identified genes are known to tion in Arabidopsis, but this regulation play roles in the stress response, but might be opposite in rice.” some have unknown functions. Rakwal To examine the effects of ozone on notes that novel transcription factors, rice, seedlings were grown for 2 weeks
MAP kinases, and calcium protein kinases were found to be up- or downregulated in ozone-treated rice. Some of the metabolism genes are involved in the production of antioxidants; this is expected because ozone causes the generation of reactive oxygen species in plants. However, in contrast to reports on the response of A. thaliana plants subjected to ozone treatment, genes encoding for salicylic acid production were not induced in rice. Rice proteins were separated by 2DE and analyzed by MS/MS. A total of 21 nonredundant proteins were identified as being differentially regulated. Rakwal points out that several fragments of RuBisCO, a protein involved in photosynthesis, were identified in ozone-treated seedlings. Thus, the protein probably was degraded into smaller fragments in response to ozone treatment. In addition, defense-related proteins were upregulated in treated plants. Protein levels and levels of the corresponding transcripts didn’t always correlate well, but Rakwal explains that, in many cases, this could be chalked up to regulation of protein activities by posttranslational modifications. In contrast, transcript levels of genes involved in metabolic pathways generally correlated well with metabolite levels. Metabolites were analyzed by CE/MS, and 24 were differentially regulated. Rakwal says that γ-aminobutyric acid (GABA), which functions as a neurotransmitter in mammals, was increased in ozone-treated rice. Although the researchers don’t know the exact role of GABA in plants, Rakwal hypothesizes that it “probably plays a dual role as both a signaling molecule and a metabolite.” Rakwal and colleagues now have several good leads in their quest to dis cover the molecular effects of ozone on rice plants. Eventually, they want to find molecular markers that could help them evaluate the tolerances of various rice cultivars. The ultimate goal is to integrate ozone data into a larger-scale climate change project that includes the study of CO2 levels and high temperature, says Rakwal. —Katie Cottingham
Journal of Proteome Research • Vol. 7, No. 7, 2008 2591
