New evidence for the presence and function of phosphoinositides (PPIs) in the chloroplast

Essential proteins involved in thylakoid formation, including chloroplast-localized Sec14-like protein (CPSFL1) and the vesicle-inducing protein in plastids (VIPP1), bind PPIsin vitrowith high affinity. PPIs are a class of phospholipids characterized by a phosphorylated inositol head group. Although PPIs constitute a relatively small proportion of the total phospholipids, they play essential roles in various regulatory processes. The exact subcellular localization of most PPI species in plants is incomplete due to their rapid turnover and low abundance. Currently there is no documented evidence for the presence and function of phosphoinositides (PPIs) in chloroplasts. In our study, we developed genetically encoded biosensors targeted into plastids, enabling the detection of various PPI isoforms (PI3P, PI4P, PI5P, PI(4,5)P₂and PI(3,5)P2) within the chloroplasts. We effectively demonstrated the specificity of our PPI biosensors in detection of PPIs, as evidenced by the alterations in PPI biosensor distribution patterns upon co-expression of PPI modifying enzymes (cTP-SAC7,cTP-PTEN, andcTP-dOCRL). Additionally, our research confirmed the capability of the generated PPI biosensors to detect PPIs within the chloroplasts of both tobacco andArabidopsisin a concentration-dependent manner. Furthermore, we unveiled the association and potential interaction of PI3P with VIPP1. We could show that the increased PPI flux within the cell during heat stress affects PPI levels in the chloroplasts, resulting in different distribution patterns of the PPI biosensors. Finally, plants expressing PPI modifierscTP-SAC7,cTP-PTEN, andcTP-dOCRL,in the chloroplast, showed increased sensitivity to drought stress, likely due to impaired PPI signaling.