PS 54-87
Small compounds targeting Arabidopsis RACK1A protein regulate diverse environmental stress resistance in crops
RACK1 (Recepter for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in eukaryotes, from plants to humans. It plays a regulatory role in diverse signal transduction and stress response pathways. Analysis of loss of function mutants in Arabidopsis indicates that RACK1A- the predominant isoform, negatively regulates environmental stress signaling, including salt stress resistance. It is hypothesized that chemical knock-out, as opposed to genetic knockout of RACK1A, will provide a functional advantage to protect plants from environmental stress. Site directed mutagenesis studies indicated that key post-translational modifications (such as sumoylation at K273 and tyrosine phosphorylation of Y248 residues) regulate RACK1A’s interaction with other proteins. In order to facilitate the identification of small compounds that bind to the functional pocket, we deduced the crystal structure of RACK1A protein at 2.4 A resolution. This crystal structure of RACK1A was used to identify dozens of small compounds that could potentially bind to the Y248 pocket. These compounds could potentially inhibit Y248 phosphorylation and bind to purified recombinant RACK1 proteins with a kD value in the micro-molar ranges. In this study, we evaluated the effectiveness of the compounds in regulating environmental stress responses in Arabidopsis thaliana and a variety of agricultural plants.
Results/Conclusions
Since it is known that RACK1 is involved signal transduction for the growth hormone, auxin, we expected that the small compounds we raised to regulate the function of RACK1 would regulate auxin. Our results show that SD29, one of the key small compounds, positively regulates the pathway of auxin in Arabidopsis. It is also known that RACK1 is involved in many stress response pathways that are mediated through the production of reactive oxidative species. Our results using a diaminobenzadine (DAB) assay indicate that the small compounds we identified as potential regulators do inhibit the generation of reactive oxidative species in Arabidopsis in response to specific stressors, like salt. These compounds are also effective in regulating salt stress responses in a wide variety of crop plants including tomato, beans, and pepper. Finding the genetic mechanisms that regulate environmental stress responses in plants has high relevance to studies of ecological genetics. This work is also important for developing plants suited for revegetation of degraded areas and agriculture in marginal environments.