Metabolomics and physiological analyses reveal β-sitosterol as an important plant growth regulator inducing tolerance to water stress in white clover.

β-sitosterol influences amino acids, carbohydrates, organic acids, and other metabolite metabolism and homeostasis largely contributing to better tolerance to water stress in white clover. β-sitosterol (BS) could act as an important plant growth regulator when plants are subjected to harsh environmental conditions. Objective of this study was to examine effects of BS on growth and water stress tolerance in white clover based on physiological responses and metabolomics. White clover was pretreated with or without BS and then subjected to water stress for 7 days in controlled growth chambers. Physiological analysis demonstrated that exogenous application of BS (120 μM) could significantly improve stress tolerance associated with better growth performance and photosynthesis, higher leaf relative water content, and less oxidative damage in white clover in response to water stress. Metabolic profiling identified 78 core metabolites involved in amino acids, organic acids, sugars, sugar alcohols, and other metabolites in leaves of white clover. For sugars and sugar alcohol metabolism, the BS treatment enhanced the accumulation of fructose, glucose, maltose, and myo-inositol contributing to better antioxidant capacity, growth maintenance, and osmotic adjustment in white clover under water stress. The application of BS was inclined to convert glutamic acid into proline, 5-oxoproline, and chlorophyll instead of going to pyruvate and alanine; the BS treatment did not significantly affect intermediates of tricarboxylic acid cycle (citrate, aconitate, and malate), but promoted the accumulation of other organic acids including lactic acid, glycolic acid, glyceric acid, shikimic acid, galacturonic acid, and quinic acid in white clover subjected to water stress. In addition, cysteine, an important antioxidant metabolite, was also significantly improved by BS in white clover under water stress. These altered amino acids and organic acids metabolism could play important roles in growth maintenance and modulation of osmotic and redox balance against water stress in white clover. Current findings provide a new insight into BS-induced metabolic homeostasis related to growth and water stress tolerance in plants.