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Spiraea fritschiana  ( S. fritschiana ) is widely cultivated in horticulture and exhibits strong cold tolerance. We identified both glutathione peroxidases (GPXs) and glutathione S-transferases (GSTs) as key proteins involved in the low-temperature response of  S. fritschiana.  Both enzymes are known to participate in reactive oxygen species (ROS) scavenging through the ascorbate—glutathione cycle. Previous experiments have demonstrated that both  SfGPX  and  SfGST  are localized in the cytoplasm and exhibit responsiveness to low-temperature induction. In this context, we hypothesize a potential functional interplay between these two genes in mediating  Spiraea ’s cold tolerance. To elucidate the functional interplay between  SfGPX  and  SfGST  in cold stress adaptation, this study employed yeast two-hybrid (Y2H) and luciferase complementation assays (LCA) to validate their protein–protein interactions, providing a foundation for further mechanistic studies. Meanwhile, we constructed  SfGPX ,  SfGST  overexpressing and  SfGPX / SfGST  co-overexpressing lines of  Nicotiana benthamiana  ( N. benthamiana ). We monitored phenotypic alterations and quantified key physiological parameters related to cold tolerance, photosynthetic performance and Ca 2+  signaling in all transgenic lines under low-temperature conditions. Transient overexpression of  SfGPX ,  SfGST  and  SfGPX/SfGST  in  Spiraea japonica  ‘Gold Mound’ ( S. japonica  ‘Gold Mound’), and transient editing of these genes in  S. fritschiana  were performed. The relative electrical conductivity (REC) was detected in each line under low temperature. The results showed that both Y2H and LCA detected no direct  SfGPX–SfGST  interaction under our experimental conditions. Under low-temperature stress conditions,  SfGPX/SfGST  co-overexpressing  N. benthamiana  lines exhibited significantly improved cold tolerance compared with single-gene overexpression lines. Co-overexpression of  SfGPX/SfGST  in  S. japonica  ‘Gold Mound’ reduced REC under cold stress versus single-gene lines, while co-edited  S. fritschiana  showed increased REC. This result suggests that while  SfGPX  and  SfGST  do not exhibit a reciprocal relationship in regulating low-temperature tolerance in tobacco and the two  Spiraea  species, their co-expression demonstrates a synergistic effect.

Co-expression of SfGPX and SfGST in Nicotiana benthamiana and two Spiraea species further regulates their low-temperature tolerance mechanism