AbstractPerennial grasses will account for approximately 16 billion gallons of renewable fuels by the year 2022, contributing significantly to carbon and nitrogen sequestration. However, perennial grasses productivity can be limited by severe freezing conditions in some geographical areas, although these risks could decrease with the advance of climate warming, the possibility of unpredictable early cold events cannot be discarded. We conducted a study on the model perennial grass Brachypodium sylvaticum to investigate the molecular mechanisms that contribute to cold and freezing adaption. The study was performed on two different B. sylvaticum accessions, Ain1 and Osl1, typical to warm and cold climates, respectively. Both accessions were grown under controlled conditions with subsequent cold acclimation followed by freezing stress. For each treatment a set of morphological parameters, transcription, metabolite, and lipid profiles were measured. State-of-the-art algorithms were employed to analyze cross-component relationships. Phenotypic analysis revealed higher adaption of Osl1 to freezing stress. Our analysis highlighted the differential regulation of the TCA cycle and the GABA shunt between Ain1 and Osl1. Osl1 adapted to freezing stress by repressing the GABA shunt activity, avoiding the detrimental reduction in fatty acid biosynthesis and the concomitant detrimental effects on membrane integrity.