Monitoring ice-binding activity during ice-affinity purification of proteins from <i>B</i>. <i>distachyon</i> leaves.

<p>The crude cell lysates (0.1 mg/mL of total protein) of non-acclimated (NA) and cold-acclimated (CA) (1 week at 4°C) leaf tissue, as well as samples collected from the ice and liquid fractions during the purification procedure were tested for ice-recrystallization inhibition activity. Samples were observed at time 0 and following an 18 h incubation period at −4°C with all assays conducted in triplicate.</p

Knockdown of Ice-Binding Proteins in <i>Brachypodium distachyon</i> Demonstrates Their Role in Freeze Protection

<div><p>Sub-zero temperatures pose a major threat to the survival of cold-climate perennials. Some of these freeze-tolerant plants produce ice-binding proteins (IBPs) that offer frost protection by restricting ice crystal growth and preventing expansion-induced lysis of the plasma membranes. Despite the extensive <i>in vitro</i> characterization of such proteins, the importance of IBPs in the freezing stress response has not been investigated. Using the freeze-tolerant grass and model crop, <i>Brachypodium distachyon</i>, we characterized putative IBPs (<i>Bd</i>IRIs) and generated the first ‘IBP-knockdowns’. Seven IBP sequences were identified and expressed in <i>Escherichia coli</i>, with all of the recombinant proteins demonstrating moderate to high levels of ice-recrystallization inhibition (IRI) activity, low levels of thermal hysteresis (TH) activity (0.03−0.09°C at 1 mg/mL) and apparent adsorption to ice primary prism planes. Following plant cold acclimation, IBPs purified from wild-type <i>B</i>. <i>distachyon</i> cell lysates similarly showed high levels of IRI activity, hexagonal ice-shaping, and low levels of TH activity (0.15°C at 0.5 mg/mL total protein). The transfer of a microRNA construct to wild-type plants resulted in the attenuation of IBP activity. The resulting knockdown mutant plants had reduced ability to restrict ice-crystal growth and a 63% reduction in TH activity. Additionally, all transgenic lines were significantly more vulnerable to electrolyte leakage after freezing to −10°C, showing a 13−22% increase in released ions compared to wild-type. IBP-knockdown lines also demonstrated a significant decrease in viability following freezing to −8°C, with some lines showing only two-thirds the survival seen in control lines. These results underscore the vital role IBPs play in the development of a freeze-tolerant phenotype and suggests that expression of these proteins in frost-susceptible plants could be valuable for the production of more winter-hardy crops.</p></div

Developmental characteristics of wild-type <i>B</i>. <i>distachyon</i> and miRBdIRI knockdown lines.

<p>Developmental characteristics of wild-type <i>B</i>. <i>distachyon</i> and miRBdIRI knockdown lines.</p

Transcript analysis of <i>BdIRI</i>s in wild-type and knockdown (miRBdIRI) plants.

<p>Transcripts were amplified for four independent lines (miRBdIRIa-d) as well as wild-type plants with PCR performed using the isoform-specific primers listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167941#pone.0167941.t001" target="_blank">Table 1B</a>. The <i>SamDC</i> transcript was used as a PCR loading reference. Assays were done in triplicate.</p

Representative ice hemisphere obtained during the ice-affinity purification of native <i>Bd</i>IRI proteins.

<p>The polycrystalline ice hemisphere shown was obtained after two rounds of ice-affinity purification using crude cell lysates of cold acclimated (1 week at 4°C) wild-type <i>B</i>. <i>distachyon</i> leaf tissue (20 g). The ice etching observed across the surface of the ice hemisphere indicates successful incorporation of IBPs. The procedure was conducted in triplicate.</p

Ion-leakage assays conducted using leaf tissue from cold acclimated wild-type <i>B</i>. <i>distachyon</i> and miRBdIRI knockdown lines.

<p>Controls were kept at 4°C (open bars) and treated samples (dark bars) were frozen to a final temperature of −10°C over a 4 h period before assayed for ion leakage (%). Error bars represent standard error of the mean with significance denoted by an asterisk and indicating p<0.005 (unpaired T-test, one-tailed). Experiments were done in triplicate (n = 10).</p

Alignment of <i>Bd</i>IRI peptides recovered by ice-affinity purification.

<p>Peptide fragments identified by LC-MS/MS were aligned against the full-length amino acid sequences of <i>Bd</i>IRI3 (A) and <i>Bd</i>IRI4 (B). Amino acid coverage is indicated in yellow. Peptide fragments recovered by LC-MS/MS are indicated by grey boxes.</p

Amino acid sequence alignment of <i>Bd</i>IRI proteins.

<p>The <i>Bd</i>IRI amino acid sequences were aligned against the sequences of three <i>L</i>. <i>perenne</i> IRI isoforms: <i>Lp</i>IRI4 (EU680851), <i>Lp</i>IRI1 (EU680848), and <i>Lp</i>IRI3 (EU680850), using ClustalW2 multiple sequence alignment tool. N-terminal signal peptides, predicted by the SignalP 4.1 server, are underlined. Conserved leucine-rich repeat motifs (LXXL, where x represents a non-conserved residue) are highlighted in grey. The ice-binding motifs NXVXG and NXVXXG are outlined in black and grey boxes, respectively. The amino acids predicted to compose one β-helical turn, as indicated by the Phyre2 algorithm are presented as different colors. A black arrow marks the location of potential cleavage sites in the aligned residues (see text). Individual columns of residues are annotated as: (*) denoting a single, fully conserved reside; (:) denoting conservation between groups of highly similar properties (scoring > 0.5 in the Gonnet PAM 250 score), and (.) denoting conservation between groups of weakly similar properties (scoring = <0.5 in the Gonnet PAM 250 score).</p

Thermal hysteresis readings for recombinant proteins (A) and crude cell lysates of plants (B).

<p>Thermal hysteresis readings for recombinant proteins (A) and crude cell lysates of plants (B).</p

Ice-binding activity of <i>Bd</i>IRI proteins produced in <i>E</i>. <i>coli</i>.

<p>Ice recrystallization inhibition activity was observed for all 7 <i>Bd</i>IRI recombinant proteins as indicated, as well as purified recombinant <i>Lp</i>AFP and buffer controls (see text). Images were captured immediately (time 0) and following an annealing period of 18 h at −4°C (A). Ice crystal morphologies were also observed for all proteins (B). Assays were conducted using 1 mg/mL of recombinant protein, in triplicate with representative photos shown.</p