γ-aminobutyric acid is associated with calcium, reactive oxygen species, and nitric oxide responses and enhanced cadmium tolerance in Salvadora persica

Background γ-Aminobutyric acid (GABA) accumulates under stress, but its association with calcium (Ca2+), reactive oxygen species (ROS), nitric oxide (NO), and thiol responses during cadmium (Cd) exposure remains unclear in medicinal shrubs.Methods Rooted Salvadora persica L. cuttings were grown in sand-soil substrate. Screening selected 50mg Cd kg−1 substrate and 0.5 mM root-applied GABA. Eleven treatments had five independent pots each. Ca2+-sensitive Fluo-3 AM and NO-associated DAF-FM DA fluorescence and H₂O₂ were assessed in separate, destructively sampled plants at five post-application times. The 0-min value was a post-application reference; probe fluorescence was not calibrated to analyte concentration. Pot means were independent units, with time treated as a between-pot factor. A core Cd × GABA × time model and a separate categorical endpoint model were specified. The exact p-values, adjusted q-values, and model diagnostics could not be verified without pot-level records. Correlation and principal component analyses were exploratory; no path model was fitted.Results Cd reduced shoot dry weight from 3.04 to 1.89 g plant−1 (37.8%) and increased malondialdehyde from 4.37 to 9.86 nmol g−1 FW (125.6%). Relative to Cd alone, GABA increased the shoot dry weight to 2.62 g plant−1 (38.6%) and reduced the malondialdehyde to 4.34 nmol g−1 FW (56.0%). GABA was associated with a 2.2-fold greater Ca2+-sensitive fluorescence response amplitude at 30 min and 2.7-fold greater NO-associated amplitude at 360 min. LaCl₃, EGTA, DPI, DMTU, and cPTIO attenuated GABA-associated protection. Inhibitor-only and rescue controls were absent, and inhibitor phytotoxicity pre-screening was undocumented. GABA also increased total phenolics from 22.77 to 26.54 mg GAE g−1 DW (16.6%) and flavonoids from 13.21 to 15.78 mg QE g−1 DW (19.5%) relative to Cd alone.Conclusion GABA was associated with improved Cd tolerance and differences in sampled fluorescence, H₂O₂, and thiol responses. These data cannot establish a continuous signaling sequence, upstream GABA action, or target-specific pharmacological effects. The attenuation is compatible with network involvement and feedback, but pathway direction and pharmacological specificity require inhibitor-only controls, rescue experiments, and independent, well-controlled temporal validation in future studies.