Live Imaging of Subcellular Phosphate Pools in Plants

Abstract

Plants modulate photosynthesis and the subsequent fate of fixed carbon throughout each day to ensure that immediate carbon needs are met and that sufficient stores are available for the coming night. Although these processes are restricted to chloroplasts they must be tightly coordinated with metabolic pathways in the surrounding cytosol and ultimately, in distant cells within nonphotosynthetic parts of the plant. Inorganic phosphate (Pi) is a key determinant of metabolic status, and I hypothesize that the transport of inorganic phosphate (Pi) between the chloroplast and cytosol integrates metabolism in these cell compartments. PHT2;1 and PHT4;4 are unrelated Pi transporters that localize to the chloroplast inner membrane. Although I predicted that these proteins would be functionally redundant, the corresponding loss-of-function mutants exhibit opposite phenotypes with respect to biomass and starch accumulation. Based on these observations, I hypothesize that PHT2;1 and PHT4;4 transport Pi in opposite directions and that these proteins provide fine-tuning of Pi levels in the chloroplast. Because no suitable analytical technique existed to address this hypothesis I developed and optimized a series of genetically encoded fluorescent Pi biosensors for use in live plants. I confirmed that these biosensors could be targeted to different cell compartments and confirmed their utility for monitoring Pi concentrations in the cytosol and plastid stroma.