Abstract
Brassinosteroids (BR) are the steroidal phytohormones, best known for their role in plant growth and enhancing tolerance to abiotic stress for the last two decades. We investigated the effects of 24-epibrassinolide (BR - 1, 2, and 3 μM) on tomato seedlings grown under 150 mM NaCl from physiological and metabolomics perspectives. A supervised multiblock orthogonal partial least squares ANOVA (AMOPLS) analysis was performed on the untargeted metabolomics data to dissect influential factors and identify discriminant metabolites. The results showed that exogenous BR recovered the impaired photosynthetic performance induced by NaCl, as observed by increased chlorophyll content and photochemical efficiency of PSII (Phi2), while reducing PhiNPQ. Moreover, the activities of the enzymes SOD, APX, and CAT increased by 30%, 55%, and 786.3%, respectively, in BR + NaCl compared to NaCl. Unsupervised and supervised statistics revealed that, while NaCl had a dominant effect on metabolic profiles, BR modulated specific pathways like amino acids, hormone crosstalk, and secondary metabolite biosynthesis. Among phenylpropanoids and nitrogen-containing compounds, the general accumulation of lignin- and glucosinolate-related metabolites in the combined BR and NaCl treatment, compared to NaCl, indicated that BR improved plant cell membrane integrity. In addition, metabolites linked to stress defense, such as proline, glycine betaine, D-sorbitol 6-phosphate, and secologanin, accumulated. The findings identified several novel metabolites, such as N-formyl-L-kynurenine and 7,8-dihydromonapterin, attributed to BR that may support the development of NaCl-tolerant tomato plants.