Characterizing key osmolytes and osmoprotectants in drought-stressed Scotch pine: a differential approach

Abstract During drought, numerous compounds accumulate in plant tissues, but their physiological roles remain unclear – they may function as osmolytes, osmoprotectants, or merely arise as by-products of stress-induced metabolic shifts. We developed an experimental approach to link accumulation patterns with specific functions, using Scots pine ( Pinus sylvestris L.) saplings subjected to water deprivation and subsequent rewatering as a model system. We monitored changes in relative water content (RWC) and osmotic adjustment dynamics, employed untargeted primary metabolite profiling for preliminary screening of compounds correlated with water status, and performed quantitative GC-MS and LC-MS analyses of selected metabolites. Major inorganic cations (K⁺, Ca²⁺, Mg²⁺) were also quantified to assess their potential roles. Our results revealed that tryptophan, valine, and lysine – though generally present in low abundance – exhibited selective accumulation under severely reduced RWC (≤ 70%), suggesting their involvement as osmoprotectants. Major organic acids, particularly shikimic acid, showed trends consistent with osmotic adjustment. Notably, neither sucrose nor inorganic cations appeared to function as primary osmolytes in this context. The proposed approach offers a viable strategy for identifying compounds involved in plant adaptation to water deficit, with potential applications in breeding programs aimed at improving drought tolerance. Highlights An approach to identify osmolytes and osmoprotectants was implemented Accumulation of Trp, Val and Lys was consistent with their role in osmoprotection Osmotic adjustment relied predominantly on organic acids than on inorganic ions Monosaccharides but not sucrose correlates with changes in needle water status