On July 1, a research team led by Peng Qin and Shigui Li from the Rice Research Institute of Sichuan Agricultural University, in collaboration with the teams of Chengbin Xiang and Linfeng Sun from the Division of Life Sciences and Medicine at the University of Science and Technology of China, published a research article entitled “Heat-triggered phospholipid flipping stabilizes plasma membrane fluidity” in the international academic journal Nature (IF 56.1).
During the study, germinated seeds were hydroponically cultured in a JIUPO plant growth chamber under the following conditions: a temperature of 28°C, 14 h of light and 10 h of darkness per day, moderate light intensity, and 65% relative humidity. When the seedlings reached 14 days of age, the light and humidity conditions were maintained, while the seedlings were transferred to a high-temperature environment at 45°C for the designated treatment duration. After treatment, the seedlings were returned to the original growth conditions and allowed to recover for at least 3 days.
The study revealed a novel mechanism by which rice rapidly stabilizes plasma membrane fluidity through phospholipid flipping, providing new theoretical evidence and valuable genetic resources for the molecular design and breeding of heat-tolerant crops.
Extreme high-temperature events are becoming increasingly frequent worldwide, posing an increasingly severe threat to global food security. Rice is the primary staple crop in China, and its level of heat tolerance directly determines final yield. Heat stress can induce plasma membrane hyperfluidization, compromising membrane structural integrity and causing ion leakage, which can subsequently trigger cell death. Previous studies have shown that plants can alleviate heat-induced damage by remodeling membrane lipid composition through transcriptional regulation; however, this defense response often requires several hours. It has remained unclear whether plant cells possess an immediate plasma membrane protection mechanism that can be activated within minutes.
The research team first identified a heat-sensitive mutant, hot1, through screening an EMS-mutagenized rice mutant library. They subsequently cloned and mapped the key gene OsALA5, which encodes a P4-ATPase-type phospholipid flippase. Genetic analyses showed that knockout of OsALA5 reduced the survival rate of rice seedlings under high-temperature stress by more than 50% and significantly impaired multiple agronomic traits throughout the entire growth cycle. In contrast, overexpression of OsALA5 markedly enhanced heat tolerance, enabling rice plants to maintain more stable seed-setting rates and yields under high-temperature conditions. Subsequent experiments further demonstrated that OsALA5 must assemble with its β-subunit OsALIS2 to form a heterodimer before it can be properly localized to the plasma membrane and perform its biological function. Loss of OsALIS2 function likewise resulted in a heat-sensitive phenotype in rice.
OsALA5 enhances heat tolerance in rice
Using a series of biochemical assays, including protein–lipid interaction analyses, yeast-based assays, and transport assays with reconstituted artificial liposomes, the researchers found that high temperature can alter the substrate preference of OsALA5 within minutes. Under normal-temperature conditions, OsALA5 preferentially binds unsaturated phosphatidylcholines (PCs). Following heat stress, however, it specifically recognizes and transports saturated phosphatidylcholines to the cytoplasmic leaflet of the plasma membrane. Leaflet-resolved lipidomics confirmed this rapid lipid redistribution. Meanwhile, short-term heat treatment did not affect OsALA5 transcript levels or protein abundance, indicating that this process represents a post-translational regulatory mechanism independent of gene expression. Structural analyses of the protein showed that high temperature induces expansion of the OsALA5 substrate-binding pocket, creating a spatial conformation more favorable for binding rigid saturated fatty-acyl chains. This provides a structural explanation for the molecular mechanism underlying the heat-induced switch in substrate preference. Measurements of membrane fluidity and physiological assays further confirmed that OsALA5 stabilizes plasma membrane structure by enriching saturated phosphatidylcholines, effectively preventing excessive plasma membrane fluidization, reducing ion leakage, and suppressing cell death. This regulatory function is also evolutionarily conserved in Arabidopsis thaliana and yeast.
The OsALA5–OsALIS2 complex exhibits enhanced binding to saturated PCs under heat stress
OsALA5 selectively transports saturated PCs to the cytoplasmic leaflet of the plasma membrane under heat stress
The researchers performed haplotype analysis of the OsALA5 promoter and coding sequences in 2,236 accessions of Asian cultivated rice and identified seven haplotypes. The rare haplotype Hap7, which accounted for only 0.09% of the population, exhibited significantly higher gene transcription levels than the other haplotypes as a result of promoter sequence variation. This haplotype is present in the heat-tolerant Aus-type rice varieties Nagina22 and Kas. When Hap7 from Kas was introduced into the Nipponbare (Nip) genetic background, which carries Hap2, the heat tolerance of the resulting rice plants was markedly enhanced. The survival rate under high-temperature stress at the seedling stage increased by nearly 42 percentage points; under heat stress during the heading and flowering stage, the seed-setting rate increased by 164%, while yield per plant increased by 135%. Multi-year, multi-location field trials demonstrated that rice carrying Hap7 showed stable improvements in seed-setting rate, thousand-grain weight, and grain yield, without adverse effects on agronomic traits such as plant number and plant height. The yield increase was particularly pronounced at the Changsha field site, where high-temperature events occur frequently. These results demonstrate that this allele can stably safeguard yield under high-temperature field conditions and has strong potential for direct application in crop breeding.
Natural variation in OsALA5 enhances heat tolerance and yield in rice
In summary, this study challenges the conventional view that heat-associated lipid remodeling in plants relies solely on transcriptional regulation. It provides the first demonstration of a rapid plasma membrane homeostasis mechanism mediated by minute-scale phospholipid flipping, thereby expanding the temporal framework of plant high-temperature responses. The study also identified a favorable rare haplotype of OsALA5 and demonstrated that it can stably increase rice seed-setting rate and yield under high-temperature conditions, providing a valuable genetic resource for molecular breeding of heat-tolerant rice.