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

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). And monitored phenotypic alterations and quantified key physiological parameters related to cold tolerance, photosynthetic performance and Ca2+ 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 to 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.