Heat-triggered phospholipid flipping stabilizes plasma membrane fluidity.

Cells must rapidly counteract heat stress-induced hyperfluidization of the plasma membrane to prevent membrane damage1,2, yet how cells achieve such early protection remains unknown. Here we show that in rice (Oryza sativa), the P4-ATPase OsALA5, together with its β-subunit OsALIS2, mediates a heat-responsive flipping of saturated phosphatidylcholines that rapidly stabilizes plasma membrane fluidity. Using leaflet-resolved lipidomics and complementary transport assays, we demonstrate that heat exposure induces a minute-timescale shift in OsALA5 transport activity that leads to selective enrichment of saturated phosphatidylcholines in the cytoplasmic plasma membrane leaflet. This OsALA5-mediated saturated phosphatidylcholine flipping prevents plasma membrane hyperfluidization upon heat stress, thus mitigating ion leakage and cell death. Our analyses of OsALA5 orthologues in Arabidopsis thaliana and yeast support functional conservation of a rapid heat-associated response within a subset of plasma membrane-localized, phosphatidylcholine-transporting P4-ATPases. We identified a rare haplotype of OsALA5 that confers both heat tolerance and yield stability in multi-year, multi-location field trials. Thus, beyond identifying this P4-ATPase-mediated flipping of saturated phosphatidylcholines in response to heat stress and providing genetic resources to advance breeding of heat-tolerant crops, our study reveals how cells counteract heat stress-driven plasma membrane hyperfluidization at an earlier stage than the previously known transcription-dependent lipid remodelling response.