Phosphorylation of PIF3 by MPK6 is required for coordinated regulation of miRNA biogenesis and hypocotyl elongation in Arabidopsis

Light is one of the most important environmental factors that affect overall growth and development in plants. PHYTOCHROME INTERACTING FACTORs (PIFs) are negative regulators of photomorphogenesis. PIFs mediate light responses by interacting with downstream molecular partners and are essential regulators for hypocotyl elongation in Arabidopsis. Light induce activation of phytochromes (phys), promotes rapid phosphorylation, ubiquitination and degradation of PIFs. However the kinase responsible for the phosphorylation of PIFs and the signaling mechanism governing the adaptive changes leading to hypocotyl elongation to differential light in-tensities is not well understood. Here, we report interaction and phosphorylation of PIF3 by a mitogen-activated protein kinase 6 (MPK6) both in vitro and in vivo. Phosphorylation was significantly abolished when all the PIF3 putative phosphorylation sites were mutated to alanine. Further, we found that PIF3 directly binds to the bHLH binding domain in the promoter of RL responsive miRNA, miR163. Interestingly, phosphorylation status of PIF3 substantially affects the binding of PIF3 and mutated proteins to miR163 promoter. Further, overexpression of PIF3 affects the expression of red light (RL) responsive miRNAs and downstream genes involved in hypocotyl elongation and seedling development. These results suggest that MPK6-PIF3 module functions upstream of RL-responsive miRNAs and PIF3-regulated genes involved in photomorphogenesis, thus interconnecting these pathways together.

Automated summary

Light is a crucial environmental factor in plant growth and development. PIFs are negative regulators involved in light responses and are essential for hypocotyl elongation in Arabidopsis. This study found that MPK6 phosphorylates and interacts with PIF3, and the phosphorylation affects its binding to miR163 promoter. Overexpression of PIF3 influences the expression of RL-responsive miRNAs and genes involved in photomorphogenesis. These results suggest that the MPK6-PIF3 module plays a role in connecting RL-responsive miRNAs and PIF3-regulated genes.