Identification of phytochrome-interacting protein candidates in Arabidopsis thaliana by co-immunoprecipitation coupled with MALDI-TOF MS

Phytochrome‐interacting proteins have been extensively studied to elucidate light‐signaling pathway in plants. However, most of these proteins have been identified by yeast two‐hybrid screening using the C‐terminal domain of phytochromes. We used co‐immunoprecipitation followed by proteomic analysis in plant cell extracts in an attempt to screen for proteins interacting either directly or indirectly with native holophytochromes including the N‐terminal domain as well as C‐terminal domain. A total of 16 protein candidates were identified, and were selected from 2‐DE experiments. Using MALDI‐TOF MS analysis, 7 of these candidates were predicted to be putative phytochrome A‐interacting proteins and the remaining ones to be phytochrome B‐interacting proteins. Among these putative interacting proteins, protein phosphatase type 2C (PP2C) and a 66‐kDa protein were strong candidates as novel phytochrome‐interacting proteins, as knockout mutants for the genes encoding these two proteins had impaired light‐signaling functions. A transgenic knockout Arabidopsis study showed that a 66‐kDa protein candidate regulates hypocotyl elongation in a light‐specific manner, and altered cotyledon development under white light during early developmental stages. The PP2C knockout plants also displayed light‐specific changes in hypocotyl elongation. These results suggest that co‐immunoprecipitation, followed by proteomic analysis, is a useful method for identifying novel interacting proteins and determining real protein‐protein interactions in the cell.