INTERACTION STUDIES OF CYTOKININ SIGNALING PROTEINS IN ARABIDOPSIS THALIANA

Abstract

Cytokinins, a major class of phytohormones, are vital in various developmental processes like cell division and differentiation. The signaling pathway is derived from the two component system (TCS) present in bacteria and fungi. The signal is perceived by AHKs (Arabidopsis Histidine kinases) on the cell membrane, transferred to AHPs (Arabidopsis Histidine Phosphotransfer Proteins) in cytosol, which then transfer it to type-B and type-A ARRs (Arabidopsis Response Regulators) in the nucleus. It is known that AHPs transfer the phosphoryl group to type-A ARRs, which in turn, get activated and regulate downstream functions. However, some of the key questions such as the molecular basis of phosphorelay from AHPs to type-A ARRs and the processes associated with the activation of type-A ARRs subsequent to phosphorylation are yet to be addressed.

In this work, we generated a homology model of AHP1-ARR4 (16-175 amino acid residues) using the crystal structure of a homologous protein complex in yeast as template. Based on the model, selected amino acid residues of ARR4 located at the interaction interface of AHP1 ¿ ARR4 were identified and mutated. Some of the mutations affected the interaction with AHP1 in yeast-two hybrid system, thereby indicating their importance in interaction. The impact of the mutations of the selected amino acid residues of ARR4 were also examined on the biological functions of ARR4 in planta by complementing the hextuple type-A ARR (arr3 arr4 arr5 arr6 arr8 arr9) knock-out background with either wild-type or mutant variants of ARR4. The various transgenic lines were examined for their cytokinin sensitivity, and the results obtained suggest a possibility of the role of strength of interaction on phosphorylation and hence protein function. Simultaneously, we found that homologs of ARR4 in bacteria, yeast and Dictyostelium have conserved Ser/Thr and an aromatic amino acid (Phe/Tyr), known as ¿the aromatic switch¿ that plays a role in the activation of proteins upon phosphorylation. The underlying mechanism involves a conformational shift of the Phe/Tyr from an outward position to an inward position (the switch) in the 3-D structure of the protein. This is facilitated by Ser/Thr in response to phosphorylation on Asp. It was further identified that ARR4 in Arabidopsis also has a similar region in the protein with the critical amino acid residues conserved. The plant data from our study clearly indicated the role of these conserved residues in the biological functions of ARR4. This highlights the possibility that the conserved residues might be playing a similar function in ARR4 as in other organisms. Hence, our data revealed a key step in the molecular mechanism of cytokinin signaling, namely, activation of the response regulator proteins upon phosphorylation.