Objective Phosphoenolpyruvate carboxylase kinase-related kinases (PEPKRs), belonging to the plant-exclusive CDPK/SnRK superfamily, have not yet been fully elucidated in terms of their regulatory mechanisms underlying the heavy metal stress response in Populus euphratica. This study investigated the role of Populus euphratica PEPKR2 during cadmium (Cd) stress to further elucidate the physiological and molecular regulatory mechanisms underlying plant responses to Cd toxicity.

Method The PEPKR2 gene was cloned from P. euphratica, and the DNAMAN was used for homologous amino acid sequence alignment, while a phylogenetic tree was constructed using Mega 7 software. Using wild-type (WT), empty vector control (VC), and PePEPKR2-overexpressing Arabidopsis thaliana lines (PePEPKR2-OE1, PePEPKR2-OE7, and PePEPKR2-OE11) as experimental materials, plants were subjected to graded cadmium treatments to investigate the mechanistic role of PePEPKR2 in plant responses to Cd stress at both physiological and molecular levels.

Result (1) The amino acid sequence of P. euphratica PEPKR2 (PePEPKR2) exhibited the highest sequence similarity and closest phylogenetic relationship with its ortholog in Populus trichocarpa. Following cadmium stress treatment, differential expression patterns of PEPKR2 were observed across root, stem, and leaf tissues in P. euphratica, with statistically significant alterations in transcript abundance. Subcellular localization assays confirmed the nuclear compartmentalization of PePEPKR2 protein. (2) Under cadmium stress conditions, PePEPKR2-overexpressing plants exhibited significantly reduced root elongation, survival rate, and fresh biomass compared with WT and VC plants, whereas relative electrolyte leakage was markedly elevated. These phenotypic divergences collectively demonstrated hypersensitivity of PePEPKR2-overexpressing lines to Cd toxicity. (3) Following cadmium stress exposure, PePEPKR2-overexpressing plants exhibited significantly accelerated Cd²⁺ influx rates and elevated Cd²⁺ accumulation in root tips compared with WT and VC plants. Furthermore, under soil-grown conditions, transgenic lines demonstrated markedly higher Cd²⁺ concentrations in both roots and leaves relative to WT and VC plants. (4) Under cadmium stress, the PePEPKR2-overexpressing plants exhibited smaller increases in superoxide dismutase and catalase activities, but greater decreases in peroxidase activity compared with WT and VC plants. These changes in antioxidant enzyme activities were consistent with the transcriptional patterns of their corresponding genes. Consequently, the PePEPKR2-overexpressing lines accumulated significantly higher levels of H₂O₂ in root tips than the controls. Under soil culture conditions, after cadmium stress treatment, overexpressed lines exhibited lower relative chlorophyll content, maximum photochemical efficiency, actual photochemical efficiency, electron transport rate, net photosynthetic rate, transpiration rate, and stomatal conductance while higher intercellular CO₂ concentration than WT and VC lines.

Conclusion Overexpression of PePEPKR2 enhances Cd²⁺ accumulation in Arabidopsis thaliana, compromises reactive oxygen species (ROS) scavenging capacity, and subsequently disrupts photosynthetic function, ultimately conferring negative regulation of Cd tolerance. This study provides theoretical support for genetic engineering approaches to improve cadmium phytoremediation potential in Populus species.