Heterosis,or hybrid vigor,is one of the most important tools in plant breeding and has previously been demonstrated for plant freezing tolerance.Freezing tolerance is an important trait because it can limit the geographical distribution of plants and their agricultural yield.Plants from temperate climates increase in freezing tolerance during exposure to low,non-freezing temperatures in a process termed ’cold acclimation’.Metabolite profiling has indicated a major reprogramming of plant metabolism in the cold,but it has remained unclear in previous studies which of these changes are related to freezing tolerance.In the present study,we have used metabolic profiling to discover combinations of metabolites that predict freezing tolerance and its heterosis in Arabidopsis thaliana.We identified compatible solutes and,in particular,the pathway leading to raffinose as crucial statistical predictors for freezing tolerance and its heterosis,while some TCA cycle intermediates contribute only to predicting the heterotic phenotype.This indicates coordinate links between heterosis and metabolic pathways,suggesting that a limited number of regulatory genes may determine the extent of heterosis in this complex trait.In addition,several unidentified metabolites strongly contributed to the prediction of both freezing tolerance and its heterosis and we present an exemplary analysis of one of these,identifying it as a hexose conjugate.