The objective of this study was to determine whether the novel osmolyte trimethylamine N-oxide （TMAO） enhances drought tolerance of Lotus japonicus and to preliminarily reveal the corresponding physiological mechanisms. L. japonicus ecotype ‘Miyakojima’ MG-20 was used in the study. Five concentrations of TMAO （0.1 mmol·L^-1， 0.5 mmol·L^-1， 1 mmol·L^-1， 5 mmol·L^-1， and 10 mmol·L^-1） and ddH₂O were sprayed on MG20 leaves daily for 7 d， plants were then immediately subjected to drought treatment for 28 d， followed by rewatering for 7 d. Based on plant phenotype， 5 mmol·L^-1 TMAO was selected as the best concentration to improve the drought tolerance of L. japonicus. To further investigate the underlying physiological mechanism， 5 mmol·L^-1 TMAO was sprayed on leaves for 7 d followed by drought tolerance evaluation. After 28 days of drought， plant height， leaf relative water content （LRWC）， chlorophyll content （Chl）， and photochemical efficiency （F_v/F_m） were significantly lower in both drought-stressed plants sprayed with ddH₂O （D-W） or TMAO （D-T） compared to well-watered controls sprayed with ddH₂O （W-W） or TMAO （W-T）. However， these parameters were significantly higher in the D-T group than in the D-W group. Concurrently， leaf relative electrolyte leakage （EL） and superoxide anion （O^2-） accumulation in leaves were significantly higher in the D-W and D-T groups compared to the W-W and W-T groups， but significantly lower in the D-T group than in the D-W group. Following 7 days of rewatering， chlorophyll content remained significantly higher in the D-T group compared to the D-W group， while no significant differences were observed in the other measured parameters between these two groups. These results indicate that foliar application of TMAO can alleviate drought-induced damage in L. japonicus， providing an effective approach for improving cultivation under drought conditions.

Lotus japonicus  /  TMAO  /  Drought stress  /  Physiological index