Qian Feng, Lu Bingbing, Sun Hao, Zhang Hui, Cheng Ziyin, Jin Zili, Xu Feifan, Liu Huimin
College of Horticulture and Landscape Architecture, Northeast Agricultural University, No. 600 Changjiang Road, Xiangfang District, Harbin 150030, China.
Guangzhou International Bio Island, Guangzhou National Laboratory, Guanzhou Road, Haizhu District, Guangzhou 510005, China.
Tree Physiol. 2025 Jul 1;45(7). doi: 10.1093/treephys/tpaf069.
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 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 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.
华北珍珠梅（Spiraea fritschiana）在园艺中广泛种植，且具有很强的耐寒性。我们鉴定出谷胱甘肽过氧化物酶（GPXs）和谷胱甘肽S-转移酶（GSTs）是参与华北珍珠梅低温响应的关键蛋白。已知这两种酶都通过抗坏血酸-谷胱甘肽循环参与活性氧（ROS）的清除。先前的实验表明，SfGPX和SfGST都定位于细胞质中，并对低温诱导有响应。在此背景下，我们推测这两个基因在介导珍珠梅耐寒性方面可能存在功能相互作用。为了阐明SfGPX和SfGST在冷胁迫适应中的功能相互作用，本研究采用酵母双杂交（Y2H）和荧光素酶互补试验（LCA）来验证它们的蛋白质-蛋白质相互作用，为进一步的机制研究奠定基础。同时，我们构建了本氏烟草（Nicotiana benthamiana）的SfGPX、SfGST过表达和SfGPX/SfGST共过表达株系。我们监测了所有转基因株系在低温条件下的表型变化，并量化了与耐寒性、光合性能和Ca2+信号相关的关键生理参数。在金山绣线菊（Spiraea japonica 'Gold Mound'）中瞬时过表达SfGPX、SfGST和SfGPX/SfGST，并在华北珍珠梅中对这些基因进行瞬时编辑。在低温下检测每个株系的相对电导率（REC）。结果表明，在我们的实验条件下，Y2H和LCA均未检测到SfGPX-SfGST的直接相互作用。在低温胁迫条件下，与单基因过表达株系相比，SfGPX/SfGST共过表达的本氏烟草株系耐寒性显著提高。在冷胁迫下，SfGPX/SfGST在金山绣线菊中共过表达使REC低于单基因株系，而在华北珍珠梅中共编辑则使REC升高。这一结果表明，虽然SfGPX和SfGST在调节烟草和两种珍珠梅的低温耐受性方面没有相互关系，但它们的共表达显示出协同效应。
