Roles of Chloroplastic Phosphate Transporters in Modulation of Photosynthesis in Arabidopsis thaliana

Abstract

Inorganic phosphate (Pi) is an essential nutrient for all living organisms, but in plants it has specialized roles in photosynthesis and the regulation of carbon metabolism. During the photoperiod, the concentration of Pi in the chloroplast stroma must be maintained within relatively narrow limits. A low concentration would impose a substrate-level limitation for ATP synthesis, whereas high Pi concentrations would allosterically inhibit starch synthesis. The contribution of Pi transport processes to the control of stromal Pi concentration is largely unknown. In this work, I used live imaging of a genetically encoded Förster Resonance Energy Transfer (FRET)-based Pi biosensor to evaluate the roles of three chloroplastic Pi transporters, TPT, PHT2;1 and PHT4;4, in stromal Pi homeostasis in the model plant Arabidopsis thaliana. I discovered that in wild-type plants, stromal Pi levels vary spatially and temporally within the four major tissues of the leaf. Analysis of loss-of-function mutants demonstrated that TPT and PHT2;1 both catalyze Pi import with overlapping but distinct tissue-specificities, whereas I found no evidence of Pi transport catalyzed by PHT4;4. I also found that tpt and pht2;1 mutations have opposite effects on cytosolic Pi accumulation, which further points to distinct physiological functions for TPT and PHT2;1. This finding also negates the long-standing view that cytosolic Pi levels are held constant through compensation by vacuolar Pi pools. I used the transporter mutants to evaluate the effects of reduced stromal Pi levels on photosynthetic processes in the thylakoid. Chlorophyll fluorescence-based measurements showed that photosynthetic efficiency correlates with stromal Pi concentration. Moreover, electrochromic shift (ECS) measurements revealed Pi-dependent regulation of ATP synthase activity that involves dynamic, compensatory modulation of the thylakoid proton motive force to alter the affinity of ATP synthase for its Pi substrate. As part of an unrelated study, I explored the relationship between lignin biosynthesis and the GPT2 plastidic glucose-6-phosphate/Pi transporter. Qualitative and quantitative analyses of the lignin composition revealed reduced lignin in an RNAi line (HCT-RNAi) that suppressed expression of a key enzyme, HCT, in the lignin biosynthetic pathway, but hyper-accumulation of lignin in a GPT2 overexpression line and a gpt2-1 mutant that was previously described as a loss-of-function mutant. Metabolomic and transcriptomic analyses showed that these opposite effects on lignin accumulation are associated with induction of stress response genes and transcriptional re-programming of the starch and sucrose metabolism pathways.