Appropriate responses to environmental stresses or developmental cues are critical for the survival of all organisms including plants. Protein kinases are enzymes that play a central role during these processes by transducing external and internal signals to downstream cellular components resulting in the modulation of plant growth accordingly. Thus, the accurate detection of kinase activity is critical for understanding the mechanism governing the plantâ€™s response to various signals, which may lead to the development of new crops with better stress resilience and productivity. The goal of this proposed study is to develop a new assay that facilitates the detection of kinase activity in plants. This project not only provides a novel approach to detect kinase activity in a facile manner but also enables the researcher to overview dynamic cellular changes in response to various stresses and growth conditions. The project will also provide research training to graduate and undergraduate students who will acquire interdisciplinary techniques integrating molecular biology and chemical analysis.
Protein kinases modulate the phosphorylation status of cellular targets, thereby regulating plant growth, immunity, and stress responses. Dysregulation of kinase activity often leads to severe defects in the overall fitness of plants and stress responses. Because of its critical roles in plants, kinase activity can be used to deduce the status of signal transduction and the plantâ€™s stress response. In this respect, kinase activity assays with high sensitivity, specificity, cost-efficiency, ease of use, or multiplexing capability are critical prerequisites for researchers to accurately measure in vivo and in vitro kinase activity. However, most current kinase assays still lack at least some desired capabilities. Emerging DNA-based activity measurements of enzymes in drug discovery research have shown promise in addressing the unmet need for optimal kinase assays in the field, providing a kinase toolbox with the desired aspects. The goal of this proposal is to develop a kinase assay, taking advantage of the strength of DNA-based enzyme activity measurement, and to validate the assay for in vivo plant kinome studies by using the plant hormone ethylene and brassinosteroid signaling as a model system. The proposed kinase assay uses DNA-encoded peptide substrates as activity probes and qPCR-based quantification of kinase activity. Given the versatility of qPCR in detecting extremely low concentrations of DNA with high specificity through unique DNA sequence-mediated barcoding for substrate peptides, the investigators hypothesize that the proposed DNA-encoded kinase assay should be able to overcome the limitations of current kinase assays and become a valuable tool in plant signal transduction research. Furthermore, by using the multiplexing capability of the assay and next-generation DNA sequencing, they will develop DNA-encoded peptide arrays that provide a simultaneous quantitative dynamic overview of the changes in cellular phosphorylation events in plants.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.