Reconstruction of the raffinose family oligosaccharide pathway in maize improves heat stress tolerance

Raffinose family oligosaccharides (RFOs), primarily raffinose and stachyose, are crucial for plant abiotic stress tolerance. Raffinose is synthesized by RAFFINOSE SYNTHASE (RAFS) from galactinol and sucrose, whereas stachyose can be produced by STACHYOSE SYNTHASE (STS) from galactinol and raffinose. While Arabidopsis possesses functional RAFS and STS genes and produces both raffinose and stachyose, maize lacks both a functional STS gene and endogenous stachyose. Here, we show that Arabidopsis STACHYOSE SYNTHASE (AtSTS) is induced by heat stress, and its overexpression and subsequent stachyose accumulation enhance heat tolerance. To extend the RFOs pathway in maize, we introduced the AtSTS expression cassette into the maize genome. The resulting transgenic maize lines accumulated stachyose and exhibited improved heat tolerance. Under heat stress, stachyose amounts in these lines increased significantly, likely due to the induction of endogenous ZmRAFS and subsequent protein and raffinose production, the latter serving as the STS substrate. Compared with wild-type controls, transgenic maize seedlings showed reduced leaf damage, higher survival, and lower ion leakage under high-temperature conditions. The application of exogenous stachyose similarly improved heat tolerance in wild-type maize, providing further evidence of its protective function. Moreover, AtSTS overexpression reduced reactive oxygen species (ROS) accumulation, indicating that stachyose helps mitigate oxidative damage. These findings establish stachyose as a contributor to heat stress resilience and propose a promising strategy for improving crop tolerance to increasing global temperatures.