Oryza sativa and Arabidopsis thaliana Scaffold Protein Receptor for Activated C Kinase 1 (RACK1) Mediated Environmental Stress Signaling Pathways

The scaffold protein, Receptor for Activated C Kinase 1 (RACK1 ), in metazoans is known to mediate many diverse signaling transduction pathways. RACK1 interacts with more then a hundred different proteins in order to regulate these signaling pathways. This highly conserved protein is present in a diverse number of species and plays a vital role in abiotic stress resistance, root and seed pod development. A combination of molecular genetic and cell biological approaches was utilized in order to uncover plant RACK1-mediated signaling pathways. RACK1 consensus sequences are found in a diverse array organisms including yeast, drosophila and humans. RACK1 studies in Oryza sativa allowed the investigation of the novel role of RACK1 in an agricultural crop. To test the functional conservation of RACK1 proteins, activation tagged rice plants over and under expressing one of the two RACK1 proteins were isolated. T-DNA and RACK1A specific primers were used to identify homozygous and heterozygous rice lines. Loss of function mutants in the model plant Arabidopsis thaliana showed hypersensitivity to the stress hormone abscisic acid (ABA). Water loss from knock-out mutants in Arabidopsis also was reduced when compared to the wild type after five hours of detachment from the plant. Under expression lines did not show stress related phenotypes and were resistant to oxidative stress. The homozygous over expression mutant rice line exhibited excessive developmental delay (plant and root development) when compared to the heterozygous and wild type rice plants. Over expression rice lines were extremely sensitive to oxidative and salt stress when compared to the wild-type. Real Time PCR based assays detected 10-12 fold more RACK1A expression in the homozygous over expression line. When treated in 60mM salt wild-type rice plants had approximately 16 fold more RACK1A expression and the over expression rice(heterozygous) has approximately 58 fold more RACK1A expression. This is evidence that salt significantly induces RACK1A gene expression. After testing antioxidant enzymes CatB, MnSOD and CuZn and the salt inducible gene P5CS all genes were down-regulated when exposed to salt and contained decreased SOD enzyme activity. Photosynthetic capacity of the heterozygous over expression rice line showed decreased photosynthetic capacity when treated in salt stress. Scaffold protein RACK1A in rice, similar to Arabidopsis, potentially can regulate environmental stress signaling pathways suggesting a strategy to identify T-DNA based RACK1A under and over expression in rice plants. With the over and under expression of rice RACK1A lines, the identification of RACK1A regulated specific developmental pathways in rice was observed and documented.