Gareth J. Warren

Freezing Sensitivity in the sfr4 Mutant of Arabidopsis Is Due to Low Sugar Content and Is Manifested by Loss of Osmotic Responsiveness

Protoplasts were tested to determine whether the freezing sensitivity of the sfr4 (sensitive to freezing) mutant of Arabidopsis was due to the mutants deficiency in soluble sugars after cold acclimation. When grown under nonacclimated conditions,sfr4 protoplasts possessed freezing tolerance similar to that of wild type, with the temperature at which 50% of protoplasts are injured (LT50) of −4.5°C. In both wild-type andsfr4 protoplasts, expansion-induced lysis was the predominant lesion between −2°C and −4°C, but its incidence was low (approximately 10%); below −5°C, loss of osmotic responsiveness (LOR) was the predominant lesion. After cold acclimation, the LT50 was decreased to only −5.6°C forsfr4 protoplasts, compared with −9.1°C for wild-type protoplasts. Although expansion-induced lysis was precluded in both types of protoplasts, the sfr4 protoplasts remained susceptible to LOR. After incubation of seedlings in Suc solution in the dark at 2°C, freezing tolerance and the incidence of freeze-induced lesions in sfr4 protoplasts were examined. The freezing tolerance of isolated protoplasts (LT50 of −9°C) and the incidence of LOR were now similar for wild type and sfr4. These results indicate that the freezing sensitivity of cold-acclimated sfr4 is due to its continued susceptibility to LOR (associated with lyotropic formation of the hexagonal II phase) and associated with the low sugar content of its cells.

Bacterialice-nucleation proteins

Certain bacteria possess proteins that enable them to nucleate crystallization in supercooled water. These ice-nucleation proteins are thought to produce templates for the assembly of very small seed crystals of ice. The proteins from different species have related, internally repetitive primary structures, which may be directly responsible for aligning the water molecules of the seed crystal.

Molecular aspects of microbial ice nucleation

Certain organisms nucleate the crystallization of ice. This requires a small volume of water to be induced, probably by lattice‐matching with a solid template, to form an‘ice embryo’—a region sharing at least some of the characteristics of macroscopic ice. It is of particular interest to understand the structure and function of biological structures capable of lattice‐matching (or otherwise inducing a quasi‐crystalline state). Some strains of the Gram‐negative eubacterial genera Erwinia, Pseudomonas, and Xanthomonas, and the mycobionts of certain lichens, display ice‐nucleating activity. In bacteria, the activity is conferred by a protein that contains three nested periodicities of repetition, which probably reflects a hierarchy of three motifs of structural repetition. Thus the tertiary structure of the ice‐nucleation protein is likely to be regular, consistent with the expectation of its forming a template for lattice‐matching. Even within a clonal culture, the nucleating sites formed by bacteria and lichens vary considerably in the threshold temperatures at which they display activity; this indicates wide variations in either the size of the template, or its structural regularity, or both. However, ice‐nucleating sites of lichen and bacterial origin are clearly differentiated by their sensitivities to experimental treatments.

Clustering of ice nucleation protein correlates with ice nucleation activity

Antibodies raised against a synthetic peptide specifically detect ice nucleation proteins from Pseudomonas species in Western blots. In immunofluorescent staining of whole bacteria, the antibodies reveal the protein in clusters, as indicated by patches of intense fluorescence in Escherichia coli cells heterologously expressing Pseudomonas ice nucleation genes. The abundance, size, and brightness of the clusters vary considerably from cell to cell. Their varying sizes may explain the variability in activity of bacterial ice nuclei. Growth at lower temperatures produces more ice nuclei, and gives brighter and more frequent patches, than growth at 37 degrees C. The observed clustering may thus reflect formation of functional ice nucleation sites in vivo. The presence of ice nucleation protein in clusters is also correlated with alterations in cell morphology.

Properties of engineered antifreeze peptides

Eight antifreeze‐like peptides were produced by cleavage from engineered chimeric proteins. One was homologous to an antifreeze peptide of the winter flounder; the others differed in length and/or sequence. The homologous peptide and all those of equal or greater length were able to inhibit recrystallization. The longer peptides were so hydrophobic that their identification required modification of the usual protocols for high pressure liquid chromatography. Their elution positions were correlated to their hydrophobicities and their lengths. Additional naturally occurring antifreezes may be identifiable with this knowledge.

Rates of assembly and degradation of bacterial ice nuclei

The kinetics of ice‐nucleus assembly from newly synthesized nucleation protein were observed following induction of nucleation gene expression in the heterologous host Escherichia coli. Assembly was significantly slower for the small proportion of ice nuclei active above ‐4.4°C; this was consistent with the belief that these nuclei comprise the largest aggregates of nucleation protein. The kinetics of nucleus degradation were followed after inhibiting protein synthesis. Nucleation activity and protein showed a concerted decay, indicating that most of the functional ice nuclei are in equilibrium with a single cellular pool of nucleation protein. A minority of the ice nuclei decayed much more slowly than the majority; presumably their nucleation protein was distinct either by virtue of different structure or different subcellular compartmentalization, or because of its presence in a metabolically distinct subpopulation of cells.

Evolutionary Perspective on the Ice Nucleation Gene-Encoded Membrane Protein

It comes as a surprise to most people that small volumes of water can be “supercooled,” that is, cooled below 0°C without freezing. The basis for this phenomenon is the requirement by most liquids for a nucleus to initiate a liquid-to-solid phase transition; liquids that are nucleus-free can supercool. The reason that supercooled water is not commonly observed is that only a single nucleus is required to initiate a freezing event, and ice nuclei are usually present in the environment. Many of these nuclei are of bacterial origin (Lindow, 1982, 1983; Warren, 1987; Warren and Wolber, 1987).