MAPKKs, as one of the main members of the mitogen-activated protein kinase (MAPK) cascade pathway, are located in the middle of the cascade and are involved in many physiological processes of plant growth and development, as well as stress tolerance. Previous studies have found that StMAPKK5 is responsive to drought and salt stress. To further investigate the function and regulatory mechanism of StMAPKK5 in potato stress response, potato variety ‘Atlantic’ was subjected to drought and NaCl treatments, and the expression of the StMAPKK5 gene was detected by qRT-PCR. StMAPKK5 overexpression and RNA interference-mediated StMAPKK5 knockdown potato plants were constructed. The relative water content, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities, as well as proline (Pro) and malondialdehyde (MDA) contents of plant leaves, were also assayed under drought and NaCl stress. The StMAPKK5 interacting proteins were identified and validated by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). The results showed that the expression of StMAPKK5 was significantly up-regulated under drought and NaCl stress conditions. The StMAPKK5 protein was localized in the nucleus, cytoplasm, and cell membrane. The expression of StMAPKK5 affected the relative water content, the enzymatic activities of SOD, CAT, and POD, and the proline and MDA contents of potatoes under drought and salt stress conditions. These results suggest that StMAPKK5 plays a significant role in regulating drought and salt tolerance in potato crop. Yeast two-hybrid (Y2H) screening identified four interacting proteins: StMYB19, StZFP8, StPUB-like, and StSKIP19. BiFC confirmed the authenticity of the interactions. These findings suggest that StMAPKK5 is crucial for potato growth, development, and response to adversity.

Sensor—responder complexes comprising calcineurin B-like (CBL) proteins and CBL-interacting protein kinases (CIPKs) are plant-specific Ca²⁺ receptors, and the CBL-CIPK module is widely involved in plant growth and development and a large number of abiotic stress response signaling pathways. In this study, the potato cv. “Atlantic” was subjected to a water deficiency treatment and the expression of StCIPK18 gene was detected by qRT-PCR. The subcellular localization of StCIPK18 protein was observed by a confocal laser scanning microscope. The StCIPK18 interacting protein was identified and verified by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). StCIPK18 overexpression and StCIPK18 knockout plants were constructed. The phenotypic changes under drought stress were indicated by water loss rate, relative water content, MDA and proline contents, and CAT, SOD and POD activities. The results showed that StCIPK18 expression was upregulated under drought stress. StCIPK18 is localized in the cell membrane and cytoplasm. Y2H shows the interaction between StCIPK18 and StCBL1, StCBL4, StCBL6 and StCBL8. BiFC further verifies the reliability of the interaction between StCIPK18 and StCBL4. Under drought stress, StCIPK18 overexpression decreased the water loss rate and MDA, and increased RWC, proline contents and CAT, SOD and POD activities; however, StCIPK18 knockout showed opposite results, compared with the wild type, in response to drought stress. The results can provide information for the molecular mechanism of the StCIPK18 regulating potato response to drought stress.

The functional role of MAPKK genes in potato (Solanum tuberosum L.) under high-temperature stress remains unexplored, despite their critical importance in stress signaling and yield protection. We characterized StMAPKK1, a novel group D MAPKK localized to plasma membrane/cytoplasm. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed cultivar-specific upregulation in potato (‘Atlantic’ and ‘Desiree’) leaves under heat stress (25 °C, 30 °C, and 35 °C). Transgenic lines overexpressing (OE) StMAPKK1 exhibited elevated antioxidant enzyme activity, including ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), mitigating oxidative damage. Increased proline and chlorophyll accumulation and reduced oxidative stress markers, hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), indicate improved cellular redox homeostasis. The upregulation of key antioxidant and heat stress-responsive genes (StAPX, StCAT1/2, StPOD12/47, StFeSOD2/3, StMnSOD, StCuZnSOD1/2, StHSFA3 and StHSP20/70/90) strengthened the enzymatic defense system, enhanced thermotolerance, and improved photosynthetic efficiency, with significant improvements in net photosynthetic rate (Pn), transpiration rate (E), and stomatal conductance (Gs) under heat stress (35 °C) in StMAPKK1-OE plants. Superior growth and biomass (plant height, plant and its root fresh and dry weights, and tuber yield) accumulation, confirming the positive role of StMAPKK1 in thermotolerance. Conversely, RNA interference (RNAi)-mediated suppression of StMAPKK1 led to a reduction in enzymatic activity, proline content, and chlorophyll levels, exacerbating oxidative stress. Downregulation of antioxidant-related genes impaired ROS scavenging capacity and declines in photosynthetic efficiency, growth, and biomass, accompanied by elevated H₂O₂ and MDA accumulation, highlighting the essential role of StMAPKK1 in heat stress adaptation. These findings highlight StMAPKK1’s potential as a key genetic target for breeding heat-tolerant potato varieties, offering a foundation for improving crop resilience in warming climates.