Objective This study investigated the physiological characteristics and differential responses of ion transport and mitogen activated protein kinase (MAPK) cascade signaling genes in Juglans cordiformes' Xinwen No.41 'walnut seedlings under different NaCl stress conditions.

Methods Pot planting was employed to explore the response of ‘Xinwen No.41’ walnut seedlings with same growth to salt stress treatments with three salt concentrations-100, 150, and 200 mmol·L⁻¹ to . , and the water treatment was served as control group (CK). Following 20 days of treatment, the phenotypic and photosynthetic parameters of the seedlings were measured. Non-invasive micro-test technology (NMT) was utilized to assess root Na⁺, K⁺, and H⁺ fluxes. RNA was extracted from both leaves and roots for gene expression analysis related to MAPK cascade signaling transduction and ion transport. Redundancy analysis (RDA) was employed to explore the correlations between physiological indicators and gene expression, aiming to elucidate the salt stress response and regulatory mechanisms in walnut seedlings.

Results Increasing salt concentrations resulted in significant reductions in root volume and total biomass of the seedlings, accompanied by leaf withering and wilting. Photosynthetic parameters such as net photosynthetic rate, transpiration rate, and stomatal conductance exhibited declining trends, while the intercellular CO₂ concentrations increased. Activities of superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX) activities, Pro, MDA and H₂O₂ contents in leaves and roots increased significantly. Na⁺ content in roots significantly accumulated and showed a clear trend of efflux with increasing salt concentration, while K⁺ efflux and H⁺ influx in leaves maintained intracellular osmotic potential homeostasis. Salt-tolerant-related genes, such as plasma membrane Na⁺/H⁺ antiporter (SOS1) exhibited higher expression levels in leaves, while the Sodium/hydrogen antiporter 1 (NHX1) was up-regulated and then down-regulated. Moreover, Vacuolar H⁺-ATPase subunit c (VHA-C1) was significantly up-regulated in roots. WRKY65 (WRKY transcription factor 65), MKK9 (Mitogen-activated protein kinase 9) and MPK3 (Mitogen-activated protein kinase 3) gene increased at 100 mmol·L⁻¹ stress, with higher expression levels observed in leaves compared to roots.

Conclusion Under NaCl stress, roots of Juglans cordiformis are more affected by stress than the leaves. JrSOS1, JrNHX1, and JrNHX2 are closely related to salt resistance and are key genes for resisting salt stress.