Objectives High content of alkaline salt is the main obstacle restricting rice growth and development in alkaline soils. Here, we compared the effects of three commonly used exogenous regulatory substances on rice seedling growth, physiological characteristics, and related gene expression under alkali stress to screen the exogenousregulatory substances that can relieve alkali stress of rice.

Methods The experiment was conducted using a hydroponic method. Zhonghua 11 was used as the test material, and exogenous regulatory substances included glycine betaine (GB), sodium nitroprusside (SNP), and melatonin (MT). Standard nutrient solution was used as control (CK), and alkaline nutrient solution with CO₃²⁻ and HCO₃⁻ concentrations of 20 mmol/L and pH 8.65 was used as alkaline stress (AS). Rice seedlings were grown for 21 days and sprayed with distilled water, GB, SNP, and MT under CK and AS conditions including eight treatments. After three days of continuous treatment, rice seedlings were harvested to determine phenotypic, physiological indexes, and gene expression levels. The relief effects of exogenous regulatory substances on rice alkaline stress were evaluated using principal component analysis and weighted comprehensive evaluation combined with membership function.

Results Compared with AS+DW, the three regulator treatments (AS+GB, AS+SNP, and AS+ MT) reduced leaf curl rate, increased seedling survival rate, and the fresh and dry weight of shoot and root of rice seedlings (P<0.05) . These treatments also decreased the content of malondialdehyde (MDA) and reactive oxygen species (H₂O₂, \rmO_\small 2^\overline \,\cdot\, ) in rice seedlings while increased the contents of proline (Pro) and soluble sugars (SS), as well as the activities of SOD, POD, and CAT. Furthermore, the expression levels of proline synthase gene OsP5CS and antioxidant enzyme synthesis genes OsCu/Zn−SOD, OsPOX1, and OsCATC were (P<0.05) induced. Under alkali stress, exogenous regulatory substances affected the expression levels of endogenous GB, NO, and MT-related synthetic enzyme genes in rice seedlings, with the SNP significantly decreasing the expression level of the OsNOA1 gene and increasing that of the OsNIA2 gene. MT (P<0.05) increased the expression level of the OsASMT1 gene and decreased the expression level of the OsSNAT gene, and GB (P<0.05) decreased the expression level of the OsBADH1 and OsCMO genes. Correlation analysis showed that OsCu/Zn−SOD was (P<0.05) positively correlated with SOD, CAT, and shoot fresh weight. Similarly, SOD was significantly and positively correlated with fresh shoot weight, and OsP5CS was positively correlated with Pro and shoot dry weight. H₂O₂ and \rmO_\small 2^\overline \,\cdot\, were negatively correlated with OsCu/Zn−SOD, SOD, and fresh shoot weight. In contrast, H₂O₂ was (P<0.05) positively correlated with leaf curl rate, while \rmO_\small 2^\overline \,\cdot\, was negatively correlated with soluble sugars (SS). Principal component analysis integrated the 16 measurement indicators into six components, with a cumulative contribution rate of 87.91%. The membership function combined with the weight method was used for a comprehensive evaluation; the D value of SNP was the largest, followed by those of GB and MT.

Conclusions GB, SNP, and MT can alleviate alkali stress and enhance rice alkali tolerance by regulating rice seedling morphology, physiological characteristics, and gene expression. The order of efficiency of alkali stress alleviation by the treatments was SNP>GB>MT.