Dynamic responses of PA to environmental stimuli imaged by a genetically encoded mobilizable fluorescent sensor

Membrane fluidity, permeability, and surface charges are controlled by phospholipid metabolism and transport. Despite the importance of phosphatidic acid (PA) as a bioactive molecule, the mechanical properties of PA translocation and subcellular accumulation are unknown. Here, we used a mobilizable, highly responsive genetically encoded fluorescent indicator, green fluorescent protein (GFP)-N160_RbohD, to monitor PA dynamics in living cells. The majority of GFP-N160_RbohD accumulated at the plasma membrane and sensitively responded to changes in PA levels. Cellular, pharmacological, and genetic analyses illustrated that both salinity and abscisic acid rapidly enhanced GFP-N160_RbohD fluorescence at the plasma membrane, which mainly depended on hydrolysis of phospholipase D. By contrast, heat stress induced nuclear translocation of PA indicated by GFP-N160_RbohD through a process that required diacylglycerol kinase activity, as well as secretory and endocytic trafficking. Strikingly, we showed that gravity triggers asymmetric PA distribution at the root apex, a response that is suppressed by PLDζ2 knockout. The broad utility of the PA sensor will expand our mechanistic understanding of numerous lipid-associated physiological and cell biological processes and facilitate screening for protein candidates that affect the synthesis, transport, and metabolism of PA.