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.