Waterlogging-Induced Changes in ABA, Carbohydrates and CBF6 Modify Freezing Tolerance in Prehardened Festuca pratensis

Changes in precipitation and snow melt during warmer winters can increase low-temperature waterlogging. Such conditions may bring about different effects when compared with a single stress trigger, such as low-temperature or water excess. The effects of waterlogging are clearly related to water temperature, and the consequences of water excess might be less severe, as more oxygen is dissolved in colder water. The effect of waterlogging during cold acclimation (CA) is poorly understood; most experiments concerning water excess are performed at relatively high-temperatures. In this study, we examined the effect of 3 weeks of waterlogging (approx. 2 cm above the soil level) on CA in Festuca pratensis Huds. (Fp), a cool-season grass. Measurements were taken before CA (after prehardening, before flooding) and after 3 weeks of CA in waterlogged (treated) and non-waterlogged (control) plants. The work included: (i) freezing tolerance test (regrowth after freezing), (ii) analysis of abscisic acid (ABA) content in the leaf, (iii) leaf stomatal conductance, (iv) leaf water content, (v) carbohydrates analysis, including fructans, and (vi) transcript levels of selected genes involved in freezing tolerance, ABA signalling and fructan biosynthesis. The aim of the study was to test a hypothesis that low-temperature waterlogging in Fp enhances freezing tolerance (plant regrowth after freezing) related to increased ABA accumulation, increased C-repeat-binding transcription factor expression and/or increased carbohydrate accumulation, including fructans. Two out of four genotypes exhibited enhanced regrowth following freezing due to waterlogging relative to control. Principal component analysis (PCA) revealed a positive correlation between ABA levels and freezing tolerance in both treatments, with a more pronounced effect observed in the waterlogged plants. However, the phytohormone played different roles in these two treatments. In the context of low-temperature waterlogging, ABA may be involved in the dehydration tolerance response in genotypes suffering from physiological drought, as well as the induction of C-repeat-binding transcription factors (CBFs) and sucrose, which may improve freezing tolerance. The increased fructan amount and polymerisation degree due to waterlogging may provide a carbohydrate sink to maintain a high photosynthetic efficiency, but are not directly responsible for freezing tolerance changes. The study indicates that tolerance mechanisms of Fp exposed to low-temperature waterlogging involve maintaining a high photosynthetic rate, as well as oxidative and dehydration stress tolerance.