Abstract
Ascorbate peroxidase (APX) is a key antioxidative enzyme which catalyzes the conversion of H2O2 to H2O and O2 in plants. In rice, APX gene family is composed of eight genes, including two in the cytoplasm (OsAPX1and OsAPX2), two in the peroxisome (OsAPX3 and OsAPX4), and four in the chloroplast (OsAPX5, OsAPX6, OsAPX7 and OsAPX8), and yet their gene regulation and functions remain mostly uncharacterized. In order to understand their responses to environmental stresses, quantitative real-time PCR (qPCR) was conducted. The results showed that most OsAPX genes were induced by NaCl, dehydration, heavy metal, and plant hormones. Analysis of the gene expression patterns of the OsAPX genes at different developmental stages indicated that OsAPX2, OsAPX3, and OsAPX8 accumulated abundant transcripts in leaf tissues at the vegetative stage. In contrast, transcripts of OsAPX1, OsAPX4, OsAPX5, OsAPX6, and OsAPX7 highly increased at the reproductive stage. In silico prediction of the 2-kb promoter region upstream of OsAPX translational start codon revealed that lots of cis-elements are related to plant hormones and abiotic stress. In order to characterize the OsAPX promoters, transcriptional start sites (TSS) of OsAPX genes were identified. In addition, 1.0 to 1.5 kb promoter region was isolated to drive GUS expression, and PAPXs/GUS transgenic rice were generated. Histochemical analysis of PAPXs/GUS plants was conducted. High activity levels of OsAPX1, OsAPX4, OsAPX5, OsAPX7, and OsAPX8 promoters were observed in root and shoot tissue of rice seedlings, and the GUS expression driven by OsAPX2, OsAPX3 and OsAPX8 promoters was accumulated mainly in the shoot tissue of rice seedlings. Most of the PAPXs/GUS plants showed GUS activity in leaf sheath, stem, and blade, but GUS activity was not found in stem tissues of both PAPX2/GUS and PAPX6/GUS plants. Strong GUS expression driven by OsAPX1, OsAPX4, OsAPX5, OsAPX7, and OsAPX8 promoters was observed in the glumes of spikelet; meanwhile, OsAPX2, OSAPX3, and OsAPX6 promoters drove GUS expression mainly in anthers. Promoter activity in mature seeds was observed in OsAPX1, OsAPX3, OsAPX4, OsAPX5, OsAPX7, and OsAPX8 genes. In summary, the diverse expression patterns of OsAPX genes indicate that OsAPX genes are induced by different environmental stresses and developmentally regulated. The analysis of PAPXs/GUS transgenic plants further indicates that OsAPX genes have tissue- and stage- specificity. This study provides basic knowledge of the regulatory OsAPX promoters, which may contribute to further functional analysis of OsAPX genes.
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