ALA3 flippase is required for adjustment of early subcellular trafficking in plant response to osmotic stress.

To compensate for their sessile lifestyle, plants developed several responses to exogenous changes. One of previously investigated and not yet fully understood adaptations occurs at the level of early subcellular trafficking, which needs to be rapidly adjusted to maintain the cellular homeostasis and membrane integrity under osmotic stress conditions. To form a vesicle, the membrane needs to be deformed, which is ensured by multiple factors, including the activity of specific membrane proteins. Here, we relate to the fact that flippases from the family of P4-ATPases actively translocate phospholipids from the exoplasmic/luminal to cytoplasmic membrane leaflet to generate curvature, which might be coupled with recruitment of proteins involved in vesicle formation at specific sites of the donor membrane. We show that lack of the ALA3 flippase activity caused defects at the PM and TGN, resulting in altered endocytosis and secretion, processes relaying on vesicle formation and movement. The mentioned cellular defects were translated into decreased intracellular trafficking flexibility failing to adjust the root growth on osmotic stress eliciting media. In conclusion, we show that ALA3 cooperates with ARF-GEF BIG5/BEN1 and ARF1A1C/BEX1 in a similar regulatory pathway of vesicle formation and together they are important for plant adaptation to osmotic stress.