Non-destructive detection of melatonin-mediated regulation of water distribution in rice seed germination under salt stress

Soil salinization has seriously threatened the national food security in saline-alkali land. Salt stress can severely inhibit rice seed germination to induce the osmotic imbalance and ionic toxicity, indicating a major constraint for rice cultivation. Melatonin (MT), an indole derivative, can be expected to enhance the salt tolerance in various crops using reactive oxygen species (ROS) scavenging and ion homeostasis regulation. However, it is lacking in specific regulations on internal water metabolism under salt stress during the seed germination stage. Since the germination is driven by water uptake and redistribution, it is often required for real-time, non-destructive monitoring of internal water status and phase transitions. Low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) techniques can be expected to non-invasively detect hydrogen proton signals, thereby precisely characterizing water content, migration, and phase distribution within biological tissues. This study aims to investigate the regulatory effects of exogenous MT on germination and internal water dynamics in rice seeds under salt stress. The research object was taken as the japonica conventional rice cultivar 'Tijin'. A preliminary screening of NaCl concentration gradient was established at 150 mmol/L as the standard salt stress condition (N150 treatment), which reduced the germination potential to 32% of the water control. The experiments included a water control group (CK1), a salt stress control group (CK2, N150), and five MT treatment groups (50, 100, 200, 400, and 800 μmol/L MT + N150). Germination physiological indices were monitored using LF-NMR (utilizing CPMG sequence) with MRI (using MSE sequence). Internal water dynamics and phase changes were tracked after the 72-hour germination period. The results revealed that there was a concentration-dependent biphasic regulation of rice seed germination by MT under salt stress. The 200 μmol/L MT concentration was identified as the optimal, significantly enhancing germination rate (94.33%), germination potential (78.67%), fresh weight accumulation, and the growth of radicles, plumules, and secondary roots. This treatment effectively alleviated salt-induced osmotic stress and ionic toxicity, indicating stable cell membrane permeability. MRI pseudo-color mapping illustrated the dynamic internal water distribution. The germination was delineated into three stages using physiological metabolism: imbibition (0-24 h), initiation (24-48 h), and germination (48-72 h). LF-NMR T₂ relaxation analysis characterized four water fractions: strongly bound water (T₂₀, 0.1-1 ms), bound water (T₂₁, 1-10 ms), semi-bound water (T₂₂, 10-100 ms), and free water (T₂₃, 100-1000 ms). Furthermore, the medium MT concentrations (200 and 400 μmol/L) effectively maintained water phase stability, indicating relaxation akin to the CK1 group. In contrast, the high MT concentration (800 μmol/L) failed to confer beneficial effects, with its water phase features. The T₂₂ peak area was consistently reduced to converge towards those of the salt-stressed CK2 group. In conclusion, an optimal concentration of exogenous MT (200 μmol/L) can effectively avoid the inhibition of salt stress on rice seed germination. There were patterns of internal water with MT-mediated alleviation of salt stress during rice seed germination. The LF-NMR and MRI techniques can be expected for the robust, non-destructive, and precise detection of the internal water dynamics and phase transitions. The finding can offer valuable theoretical and technical reference for rice seed salt tolerance.