Expression of the Cyanidioschyzon merolae stromal ascorbate peroxidase in Arabidopsis thaliana enhances thermotolerance

The ability of the primitive red alga Cyanidioschyzon merolae to adapt to high temperatures was utilized to produce thermotolerant transgenic plants. C. merolae inhabits an extreme environment (42°C, pH 2.5) and the nuclear, mitochondrial, and plastid genomes have been sequenced. We analyzed expressed sequence tag (EST) data to reveal mechanisms of tolerance to high temperatures. The stromal ascorbate peroxidase (CmstAPX) that scavenges reactive oxygen species (ROS) was expressed at high levels (4th of 4,479 entries), thus, it offers clues to understanding high-temperature tolerance. CmstAPX has a chloroplast transit peptide (cTP) and a peroxidase domain. The peroxidase domain of CmstAPX has deletions and insertions when compared with that of Arabidopsis thaliana stromal APX (AtstAPX). To clarify aspects of tolerance to oxidative and high-temperature stress, we produced transgenic A. thaliana plants overexpressing CmstAPX and AtstAPX. CmstAPX plants showed higher activities of soluble APX than those of wild-type and AtstAPX plants. Fluorescence signals of a GFP fusion protein, immuno-fluorescence, and immunogold electron microscopy showed that CmstAPX was localized in the stroma of chloroplasts. Compared with wild-type plants and AtstAPX plants, CmstAPX plants were more tolerant to oxidative stress induced by methylviologen (MV, 0.4 μM) and high-temperature stress (33°C). CmstAPX plants retained the highest chlorophyll content when treated with MV and high temperature, and their stroma and chloroplasts remained intact in their chloroplasts, whereas they disintegrated in wild-type plants. Our results suggest that the increased activity of APX in the chloroplasts of CmstAPX plants increased thermotolerance by increasing ROS-scavenging capacity at high temperatures.