Dimethyl-labelling based quantitative and functional phosphoproteomics study of blue light-induced phototropic response in Arabidopsis thaliana

Abstract

Plants are sessile organism which have developed numerous adaptive strategies to sense and orientate growth towards different stimuli. Phototropism, the directional growth of plant toward light, is instrumental for plants to develop and survive effectively. In Arabidopsis, phototropin 1 (Phot1) and phototropin 2 (Phot2) are two major blue light photoreceptors that mediate phototropism. Light-activated phototropin phosphorylation is known to be the fundamental step for phototropin signaling, yet, only a few of downstream effectors of phototropins and blue light-mediated signaling components have been discovered. Given that protein phosphorylation is one of the most abundant post translational modifications in various cellular signaling processes, we have employed a dimethyl-labeling-based 4C quantitative and functional phosphoproteomics analysis on phot1phot2 double mutant and wild type Arabidopsis, which includes Chemical labelling, Chromatographic fractionation and affinity enrichment, Computational analysis of mass spectrograms and Confirmation of PTM proteomics results by functional validation, to profile the phosphoprotoemic changes in response to a short term blue light treatment. In total, we have identified 6995 unique, repeatable and label independent phosphopeptides, corresponding to 3062 protein groups. Blue light significantly regulated 74 phosphorylation sites that are phototropin-specific, among which 56 are up-regulated and 16 are down-regulated. A member of uncharacterized protein from protein phosphatase 2C (PP2C) superfamily, which we named BLEPH1 (Blue Light Enhanced Phosphoprotein 1), exhibited impaired phototropic phenotype when it is mutated by T-DNA insertional mutagenesis. In addition, phosphorylation of BLEPH1 at S85 residues is light dependent and is directed by both phot1 and phot2. Complementation of N terminal of BLEPH1 into loss of function bleph1 mutant background rescued the defect in phototropism, conferring that PP2C phosphatase domain play a positive role in phototropism. Enhanced defect of phototropism and abnormal directional growth orientation in double mutation of BLEPH1 and its homolog, AT3G06270, further confirmed that PP2C phosphatase of BLEPH1 is critical to promote phototropic functionality. Our present study here strongly demonstrated that BLEPH1 may be an important downstream regulator of phototropin to transduces light signal from phototropin to precede phototropic response.

Date of Award	2022
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Ning LI (Supervisor)