The Lipid Flippases ALA4 and ALA5 Play Critical Roles in Cell Expansion and Plant Growth

Aminophospholipid ATPases (ALAs) are lipid flippases involved in transporting specific lipids across membrane bilayers. Arabidopsis () contains 12 ALAs in five phylogenetic clusters, including four in cluster 3 (ALA4-ALA7). / and /, are expressed primarily in vegetative tissues and pollen, respectively. Previously, a double knockout of / was shown to result in pollen fertility defects. Here we show that a double knockout of / results in dwarfism, characterized by reduced growth in rosettes (6.5-fold), roots (4.3-fold), bolts (4.5-fold), and hypocotyls (2-fold). Reduced cell size was observed for multiple vegetative cell types, suggesting a role for ALA4/5 in cellular expansion. Members of the third ALA cluster are at least partially interchangeable, as transgenes expressing in vegetative tissues partially rescued mutant phenotypes, and expression of transgenes in pollen fully rescued mutant fertility defects. ALA4-GFP displayed plasma membrane and endomembrane localization patterns when imaged in both guard cells and pollen. Lipid profiling revealed rosettes had perturbations in glycerolipid and sphingolipid content. Assays in yeast revealed that ALA5 can flip a variety of glycerolipids and the sphingolipid sphingomyelin across membranes. These results support a model whereby the flippase activity of ALA4 and ALA5 impacts the homeostasis of both glycerolipids and sphingolipids and is important for cellular expansion during vegetative growth.