Proline accumulation is inhibitory to Arabidopsis seedlings during heat stress

The effect of proline (Pro) accumulation on heat sensitivity was investigated using transgenic Arabidopsis (Arabidopsis thaliana) plants ectopically expressing the Δ(1)-pyrroline-5-carboxylate synthetase 1 gene (AtP5CS1) under the control of a heat shock protein 17.6II gene promoter. During heat stress, the heat-inducible expression of the AtP5CS1 transgene was capable of enhancing Pro biosynthesis. Twelve-day-old seedlings were first treated with heat at 37 °C for 24 h to induce Pro and then were stressed at 50 °C for 4 h. After recovery at 22 °C for 96 h, the growth of Pro-overproducing plants was significantly more inhibited than that of control plants that do not accumulate Pro, manifested by lower survival rate, higher ion leakage, higher reactive oxygen species (ROS) and malondialdehyde levels, and increased activity of the Pro/P5C cycle. The activities of antioxidant enzymes superoxide dismutase, guaiacol peroxidase, and catalase, but not those of glutathione reductase and ascorbate peroxidase, increased in all lines after heat treatment, but the increase was more significant in Pro-overproducing seedlings. Staining with MitoSox-Red, reported for being able to specifically detect superoxide formed in mitochondria, showed that Pro accumulation during heat stress resulted in elevated levels of ROS in mitochondria. Interestingly, exogenous abscisic acid (ABA) and ethylene were found to partially rescue the heat-sensitive phenotype of Pro-overproducing seedlings. Measurement of ethylene and ABA levels further confirmed that these two hormones are negatively affected in Pro-overproducing seedlings during heat stress. Our results indicated that Pro accumulation under heat stress decreases the thermotolerance, probably by increased ROS production via the Pro/P5C cycle and inhibition of ABA and ethylene biosynthesis.