Early detection and genetic insights into salt tolerance in rice by linking physiology and spectral reflectance during seed germination

Soil salinization threatens global agriculture, and seed germination is especially salt-sensitive. Although seed imbibition is a critical early phase, salt responses during this stage remain poorly understood. Reliance on final morphological traits limits nondestructive early phenotyping, thereby hindering efficient screening and mechanistic insight. To overcome this limitation, we integrated continuous physiological measurements with hyperspectral data to model dynamic responses of key indicators and further leveraged hyperspectral-derived traits for early prediction and genetic dissection of salt tolerance. The results showed that: (1) Salt stress induced a distinct temporal cascade of physiological changes during seed imbibition. Proline accumulation was the earliest response, initiating at 6 h, followed by marked increases in abscisic acid (ABA) and malondialdehyde (MDA) by 12 h, while gibberellin (GA) levels and α-amylase activity were strongly suppressed by 24 h. (2) Integrating hyperspectral reflectance with PLSR and principal component analysis (PCA) enabled high-throughput, real-time physiological monitoring and nondestructive early salt-tolerance classification. PLSR simultaneously estimated five physiological traits (R² = 0.51-0.57), which were synthesized via PCA into a comprehensive salt-tolerance score. Across time points, this pipeline achieved 94.4% cumulative accuracy by 48 h, with 55.6% of tolerant germplasms identified within 24 h, establishing a critical early diagnostic window and offering nearly a one-week lead over traditional morphological assessments. (3) Hyperspectral-derived physiological traits exhibited heritability comparable to morphological traits, and allowed the dissection of temporally resolved genetic architectures. Using the FarmCPU model, we identified 500 significant loci associated with early salt-stress responses, characterized by a complete temporal differentiation between the 24-h and 48-h stages. Allelic variation at key loci (vg0223423016, vg0712463109, and vg0910185858) significantly shaped seedling salt tolerance. Candidate gene analysis prioritized regulators involved in osmoregulation and signaling, including two aquaporin homologs (LOC_Os02g39630 and LOC_Os07g22224) and a WAK-like kinase (LOC_Os09g16550). Together, these results establish the potential of imbibition stage physiological responses for resolving final germination salt tolerance, and demonstrate that hyperspectral analysis provides a reliable technological platform to accelerate this line of research.