Genetic variation in the aquaporin TONOPLAST INTRINSIC PROTEIN 4;3 modulates maize cold tolerance

Summary Cold stress is a major abiotic stress that threatens maize ( Zea mays L.) production worldwide. Understanding the molecular mechanisms underlying cold tolerance is crucial for breeding resilient maize varieties. Tonoplast intrinsic proteins (TIPs) are a subfamily of aquaporins in plants. Here, we report that TIP family proteins are involved in maize cold tolerance. The expression of most TIP genes was responsive to cold stress. Overexpressing TIP2;1 , TIP3;2 or TIP4;3 reduced the cold tolerance of maize seedlings, while loss‐of‐function mutants of TIP4;3 exhibited enhanced cold tolerance. Candidate gene‐based association analysis revealed that a 328‐bp transposon insertion in the promoter region of TIP4;3 was strongly associated with maize cold tolerance. This transposon insertion conferred cold tolerance by repressing TIP4;3 expression through increased methylation of its promoter region. Moreover, TIP4;3 was found to suppress stomatal closure and facilitate reactive oxygen species (ROS) accumulation under cold stress, thereby inhibiting the expression of cold‐responsive genes, including DEHYDRATION‐RESPONSIVE ELEMENT BINDING FACTOR 1 ( DREB1 ) genes and a subset of peroxidase genes, ultimately attenuating maize cold tolerance. This study thus elucidates the mechanism underlying TIP‐mediated cold tolerance and identifies a favourable TIP4;3 allele as a potential genetic resource for breeding cold‐tolerant maize varieties.