The thylakoid- and pyrenoid-localized phosphate transporter PHT4-9 is essential for photosynthesis in Chlamydomonas

Phosphate (Pi) is essential for photosynthesis in the chloroplast of algae and plants. Pi homeostasis in the chloroplast is maintained by transporters from several families, whose identities in algae are largely unknown as compared with land plants. Here, we assess the role of the putative PHOSPHATE TRANSPORTER 4-9 from Chlamydomonas reinhardtii (CrPHT4-9) in maintaining chloroplast Pi homeostasis and modulating photosynthesis. Based on phylogenetic analyses and heterologous expression in a yeast (Saccharomyces cerevisiae) strain lacking Pi transporters, we demonstrate that CrPHT4-9 is a Pi transporter closely related to the chloroplast members of the PHT4 family in Arabidopsis (Arabidopsis thaliana). CrPHT4-9 is localized within the chloroplast, more specifically in the thylakoid membrane network and the tubules traversing the CO2-fixing pyrenoid. Two mutants lacking CrPHT4-9 (Crpht4-9) exhibit defective photoautotrophic growth, altered cell morphology and chloroplast ultrastructure under CO2-limiting conditions. In the Crpht4-9 mutants, we further show an increased proton motive force across the thylakoid membrane, enhanced energy- and state-transition-dependent non-photochemical quenching of chlorophyll a fluorescence, and diminished photosynthetic electron transport and ATP synthase activity. The Crpht4-9 mutants exhibit reduced affinity to inorganic carbon, indicating an impaired carbon-concentrating mechanism. These phenotypes are largely recovered by genetic complementation as well as by ample CO2 supply and, interestingly, by Pi deprivation. Therefore, we conclude that the thylakoid- and pyrenoid-localized CrPHT4-9 maintains Pi homeostasis within the chloroplast and is essential for photosynthesis and growth.