Dissecting freezing stress responses in perennial grass timothy (Phleum pratense L.) using cultivars with different adaptation backgrounds
The sustainable production of perennial grasses in Northern Norway is at risk due to the ongoing climate change. The predicted increase in temperatures and variable weather patterns are further expected to create challenges for winter survival of timothy.  Therefore, knowledge about the molecular mechanisms underlying freezing tolerance is crucial for developing robust cultivars. The current study is aimed at identifying genes involved in the freezing stress response of Timothy and studying gene expression differentiation due to field selection in contrasting environments. Four timothy cultivars, namely Engmo, Noreng, Grindstad and Snorri were field tested for three years at locations Tromsø and Vesterålen in Northern Norway. The surviving material from the field tests along with plants raised from the original seed lots were subjected to freezing tests. Following the tests, crown tissue was collected for RNA extraction. All samples from different treatment points were paired-end sequenced at 20 million reads/sample. LT50 values varied across cultivars and materials. Plant material from cold-acclimated (CA) samples was used as controls, while two pools for each cultivar were made of plant material from temperature points below the LT50, one with the higher temperatures termed Treatment 1 (T1) and one with the lower temperatures termed Treatment 2 (T2). Many genes coding for transcription factors and proteins that are known to play an important role in freezing tolerance like dehydrins, c-repeat binding factors, late embryogenesis abundant proteins are upregulated with decreasing temperatures. Moreover, genes associated with glycolysis/gluconeogenesis, TCA cycle, glutathione metabolism, proteasome, glyoxylate and dicarboxylate metabolism pathways, and genes encoding heat shock proteins, autophagy-related, plasma membrane-associated proteins, sugar and amino acid transporters had elevated expression in field survivors compared to plants raised from the original material. These DEGs might be crucial for the winter survival of timothy in a changing climate. Furthermore, differences in freezing stress response between northern and southern adapted cultivars, and surviving material from two field trial locations are discussed briefly