Light-dependent phosphorylation of THRUMIN1 regulates its association with actin filaments and 14-3-3 proteins

Light-dependent chloroplast movements in leaf cells contribute to the optimization of photosynthesis. Low-light conditions induce chloroplast accumulation along periclinal cell surfaces, providing greater access to available light, whereas high light induces movement of chloroplasts to anticlinal cell surfaces, providing photodamage protection and allowing more light to reach underlying cell layers. The THRUMIN1 protein is required for normal chloroplast movements in Arabidopsis (Arabidopsis thaliana) and has been shown to localize at the plasma membrane and to undergo rapid light-dependent interactions with actin filaments through the N-terminal intrinsically disordered region (IDR). A predicted WASP-Homology 2 domain was found in the IDR but mutations in this domain did not disrupt localization of THRUMIN1:YFP to actin filaments. A series of other protein truncations and site-directed mutations of known and putative phosphorylation sites indicated that a phosphomimetic mutation (serine to aspartic acid) at position 170 disrupted localization of THRUMIN1 to actin filaments. However, the phosphomimetic mutant rescued the thrumin1-2 mutant phenotype for chloroplast movement and raises questions about the role of THRUMIN1's interaction with actin. Mutation of serine 146 to aspartic acid also resulted in cytoplasmic localization of THRUMIN1:YFP in Nicotiana benthamiana. Mutations to a group of putative zinc-binding cysteine clusters implicate the C-terminus of THRUMIN1 in chloroplast movement. Phosphorylation-dependent association of THRUMIN1 with 14-3-3 KAPPA and OMEGA were also identified. Together, these studies provide insights into the mechanistic role of THRUMIN1 in light-dependent chloroplast movements. Site-directed mutagenesis of THRUMIN1 revealed sites critical to its blue-light-dependent localization to actin filaments, to 14-3-3 proteins, and for its regulation of chloroplast movement.

Automated summary

This study provides insights into the mechanistic role of THRUMIN1 in light-dependent chloroplast movements, including its interactions with actin filaments, the impact of phosphorylation sites, association with 14-3-3 proteins, and the involvement of THRUMIN1's C-terminus in chloroplast movement.