Elucidation of molecular basis of dehydration-induced responses aids understanding of plant adaptation and has direct implications towards fortification of sustainable agriculture. Previously, we successfully established dehydration-responsive proteome map of nucleus (1) and cell wall (2) in rice. Next we focused on mitochondria, a dynamic microenvironment for integration of cellular metabolism and signalling towards understanding energy metabolism under water-deficit conditions.
Four-week-old rice seedlings were subjected to progressive dehydration, and integrity and purity of mitochondrial fraction was evaluated. Protein identification was carried out at 1% FDR. Statistical analyses were performed on the data with a one-way analysis of variance (ANOVA) and Duncan's Multiple Range test (DMRT) using GraphPad Prism V 7.00. Gene regulation in the native system was carried out under the control of dehydration-responsive rd-29 promoter.
A critical screening of mitochondrial dehydration-responsive proteins revealed the presence of a DUF2488 or YCF54 domain containing protein, reported so far in the chloroplasts of photosynthetic eukaryotes. Stress-induced accumulation of YCF54 protein in mitochondria was confirmed by immunoblot analysis. Proteome-scale interactome networks coupled with yeast two-hybrid screening and co-immunoprecipitation analysis identified reducing enzyme, peroxiredoxin, as an interacting partner. To further characterize its function in vivo, we generated transgenic rice overexpressing the sensor under the control of rd-29 promoter. The overexpressing seedlings displayed enhanced tolerance to oxidative stress, possibly through peroxiredoxin-modulated ROS detoxification. Stress-induced overexpression of the sensor helped maintain mitochondrial respiration and ATP production. To confirm that the increased ROS detoxification is associated with mitochondrial redox homeostasis, we overexpressed both the interacting partners in non-photosynthetic eukaryote Saccharomyces cerevisiae. Interestingly, co-expression of the interacting partners showed accelerated ROS catabolism.
The present study ascertained a comprehensive view of effects of dehydration on mitochondria of rice. This study suggests that OsDUF1 plays a key role in mitochondrial redox metabolism, and confers stress tolerance.