Early Heat Shock Protein Response and Selection of Reference Genes in Arabidopsis thaliana Seedlings Subjected to Marine Fuel Contamination

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Strategies for management of damaged environments can benefit from understanding of how early petrochemical pollution affect living organisms. One of the general responses to environmental stress in plants is mediated by the regulatory network of heat shock proteins (HSP). Arabidopsis thaliana is a model plant for genetic studies, and laboratory experiments with this species might be informative for predicting analogous responses to toxicants in other species. Here, Arabidopsis seedlings were exposed to time-varying contamination (up to 24 h) with the water soluble fraction of MF380 marine fuel (WSF-MF380). An accurate estimation of expression differences in HSP genes was obtained by real-time quantitative polymerase chain reaction (qPCR). After a thorough selection and validation of reference genes, two gene pairs were found to be stably expressed across control and WSF-MF380-treated samples and were used as normalization factors. Next, we evaluated the normalized expression of five HSP genes in response to the time-varying WSF-MF380 contamination. Four HSPs presented a significant increase in gene expression, which suggests that they might be tested as biomarkers for early exposure to petrochemical compounds. While a nearly immediate response (3 h after contamination) was found for HSP90.1 and two small HSP genes localized in the mitochondria (sHSP23.5 and sHSP26.5), a slightly later response (20 h) was observed for a third small HSP with a cytoplasmic/nuclear localization (sHSP18.2). Overall, these expression changes suggest the existence of a genetic cross-talk between canonical regulatory networks of HSPs and the cellular response to non-heat stress factors, such as marine oil contamination.