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

Essential proteins such as the chloroplast-localized Sec14-like protein (CPSFL1) and the vesicle-inducing protein in plastids (VIPP1) have a high affinity for binding phosphoinositides (PPIs) in vitro. PPIs are a specific class of phospholipids characterized by a phosphorylated inositol head group, and while they make up a small fraction of total phospholipids, they are crucial for various regulatory functions. However, the precise subcellular localization of most PPI species in plants remains unclear 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 to plastids, allowing for the detection of several PPI isoforms, including PI3P, PI4P, PI5P, PI(4,5)P₂, and PI(3,5)P₂within chloroplasts. We demonstrated the specificity of these biosensors through immunological methods and observed changes in their distribution patterns when co-expressed with PPI-modifying enzymes (cTP-SAC7, cTP-PTEN, and cTP-dOCRL). Our findings also revealed the association and potential interaction between PI3P and VIPP1. Importantly, we found that elevated PPI levels during stress conditions led to altered biosensor localization, and plants expressing PPI modifiers showed increased sensitivity to drought stress, highlighting the role of PPIs in plant stress responses.