Characterization and Knockdown of a Family of Ice-Binding Proteins from Freeze-Tolerant Grasses

Abstract

Exposure to sub-zero temperatures puts plants at risk of freeze-induced damage associated with ice crystal formation causing cellular dehydration, plasma membrane rupture and cell death. Some freeze-tolerant plants induce expression of ice-binding proteins (IBPs), which adsorb to ice crystals and restrict their growth. IBPs can also result in the super-cooling of the apoplast and a modest freezing point depression. Seven IBPs (BdIRI1-7) from the annual brome grass, Brachypodium distachyon, shaped ice, restricted ice crystal growth, and exhibited low levels of freezing point depression (~0.1 °C at 1 mg/mL). Analysis of the ice-binding domain of one isoform (BdIRI1), suggested it folded as a right-handed beta-helix with two relatively flat ice-binding faces (IBFs). Site-directed mutagenesis verified that both faces were required for ice crystal adsorption, with one face acting as the primary IBF. BdIRI1 also significantly depressed the ice nucleation activity of Pseudomonas syringae extracts, suggesting a dual-function role for ice recrystallization inhibition and anti-pathogenesis. The first knockdown of ice-binding activity from any organism was achieved in B. distachyon using a pan-isoform microRNA construct. Electrolyte leakage assays showed that genetically-engineered knockdown lines had 13-22% more membrane damage than wild-type plants following freezing to −10 °C. Knockdown lines also showed trends for reduced whole-plant freezing survival following −8 °C treatments, with two lines showing significant decreases. The localization of IBPs, as well as their ability to confer freeze tolerance to a susceptible host species, was examined by the expression of IBPs from the ryegrass, Lolium perenne in Arabidopsis thaliana. Two isoforms, LpIRI2 and LpIRI3, were localized to the apoplast with the former lacking a signal peptide and presumably being secreted through a non-classical pathway, while LpAFP, an engineered construct with no signal peptide, remained intracellular. Apoplastically-localized IBPs conferred freeze resistance with a significant 13-29% decrease in electrolyte leakage and increased freeze survival at temperatures as low as −8 °C. Expression of multiple isoforms further enhanced survival indicating that IBPs might provide additive freeze protection. Together, this thesis highlights the importance of IBPs in the freezing-stress response and their potential utility in the generation of future cold-hardy crop species.