FIGURE 3.

Representative ice recrystallization inhibition “splat” assays of apoplast extracts using non‐acclimated (NA) and cold‐acclimated (CA) Brachypodium distachyon Bd21 wild type and temporal antifreeze protein (AFP) knockdown lines prOmiRBdIRI‐1e (Kd1) and prOmiRBdIRI‐3c (Kd2). Samples were annealed at −6°C for 18 h at a standardized concentration of 0.01 mg ml⁻¹. Buffer and recombinant purified rye grass Lolium perenne AFP (LpAFP) controls are also shown. Assays were performed in triplicate with similar results, and representative images are shown. Scale bars represent 0.5 mm. Splat assays performed using CA prOmiReGFP plants resulted in results similar to those obtained using wild type Bd21, indicating that the presence of the plasmid did not affect AFP activity (not shown).