Abstract
Summary 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.
Highlights
Many overwintering temperate plants are susceptible to freeze injury during the coldest months
We were interested in determining whether LpIRI2 has retained ice-binding activity and function in planta, despite the loss of the N-terminal signal peptide. We have addressed these outstanding concerns through the expression of various LpIRIP isoforms in A. thaliana, alone and in combination, in order to provide insight into the mechanisms underlying ice-binding proteins (IBPs)-mediated freezing tolerance
An Nterminal signal peptide was identified for LpIRI1, LpIRI3 and LpIRI4; as previously reported (Sandve et al, 2008), there is no apparent secretion signal within LpIRI2 or LpAFP amino acid sequences
Summary
Many overwintering temperate plants are susceptible to freeze injury during the coldest months. In order to better adapt to freezing temperatures, some overwintering plants induce the expression of a family of protective proteins, designated ice-binding proteins (IBPs). IBPs are members of a highly diverse family of proteins that have been identified in certain organisms including fish (Davies and Hew, 2013), insects (Duman, 2001), bacteria (Gilbert et al, 2004) and plants (Sidebottom et al, 2000). While freeze-avoidant organisms produce IBPs that depress the freezing point by several degrees, often referred to as antifreeze proteins (AFPs) (Davies and Hew, 2013; Duman, 2001), plants encode IBPs with low TH activity and rely on restricting ice crystal growth as a primary survival strategy (Sandve et al, 2011)
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