Ice‐binding proteins confer freezing tolerance in transgenic Arabidopsis thaliana

Lolium perenne is a freeze‐tolerant perennial ryegrass capable of withstanding temperatures below −13 °C. Ice‐binding proteins (IBPs) presumably help prevent damage associated with freezing by restricting the growth of ice crystals in the apoplast. We have investigated the expression, localization and in planta freezing protection capabilities of two L. perenne IBP isoforms, Lp IRI2 and Lp IRI3, as well as a processed IBP (Lp AFP). One of these isoforms, Lp IRI2, lacks a conventional signal peptide and was assumed to be a pseudogene. Nevertheless, both LpIRI2 and LpIRI3 transcripts were up‐regulated following cold acclimation. Lp IRI2 also demonstrated ice‐binding activity when produced recombinantly in Escherichia coli. Both the Lp IRI3 and Lp IRI2 isoforms appeared to accumulate in the apoplast of transgenic Arabidopsis thaliana plants. In contrast, the fully processed isoform, Lp AFP, remained intracellular. Transgenic plants expressing either LpIRI2 or LpIRI3 showed reduced ion leakage (12%–39%) after low‐temperature treatments, and significantly improved freezing survival, while transgenic LpAFP‐expressing lines did not confer substantial subzero protection. Freeze protection was further enhanced by with the introduction of more than one IBP isoform; ion leakage was reduced 26%–35% and 10% of plants survived temperatures as low as −8 °C. Our results demonstrate that apoplastic expression of multiple L. perenne IBP isoforms shows promise for providing protection to crops susceptible to freeze‐induced damage.