How Can it be Frozen and Still Live?
A sturdy poetic metaphor for morning frost on grass is it being as dusted in sugar. This is a prescient analogy, given the data that is emerging about how organisms that survive the low temperatures, and desiccation.
How some living organisms can survive harsh cold or drought for extended periods, where others expire with only a modest drop in temperature or water availability, has been a puzzling question in biology for some time. There are several surprising examples–compare the story of some seeds that were found in the pages of a diary that had not been touched in the 17th century that were germinated and grown successfully at Kew Gardens, with a variety of fruiting plants that are susceptible to frost and need special attention when colder weather approaches. Summer fruit like plums are amongst the most susceptible.
There are also rather ordinary examples. A surprising number of the seeds with which we are familiar, are desiccated–sunflower, rape, mustard, hemp, sesame, poppy, hazelnut, walnut, acorn, and so on. The inescapable question is how they survive being put into what amounts to a static existence. Further questions come to mind almost immediately–how do cells in stasis know when to re-animate, how do they go about doing so once they have started? In order for this process to work, a number of individual cells must work in a co-ordinated way so that the organism can bounce back into active life in a controlled way.
The first question, of how being put into dry or frozen stasis without causing death, it is a good question from a molecular perspective as well as an ecological one. Lipids, and thus cell membrane studies, are central to this. We know from lipid physics studies that when the temperature falls below a certain point, the packing of the alkyl fraction of lipid bilayers changes in a way that heavily compromises its barrier properties. Significant drops in temperature can crystallise membranes.
A recent paper by Konov et al.  gives us some clues as to how scientists are beginning to understand resistance to these changes in lipid packing. In some recent work , they investigate the mediating effect of sugars (e.g. sucrose, trehalose) and sugar alcohols (e.g. sorbitol) on the behaviour of model lipid bilayers. They showed that these sorts of molecules stabilise the bilayers we see under ambient conditions, so that they remain in place at lower temperatures.
Figure. Trehalose (1, top left), surose (2, middle left) and sorbitol (3, bottom left), trehalose dibehenate (4, right).
What this group of authors conclude is that these sugars have the ability to replace the water molecules that interact with the bilayer under more ambient conditions. The molecular structure of the water and sugars are a clue to this (Figure). This means that the membrane behaves much more like it would do when water is present. This in turn has led to such molecules being called ‘cryoprotective’ reagents.
There are several biophysical techniques that could have be used to test the hypothesis that sugars are cryoprotective agents. In principle, any biophysical technique that can be used to indicate transitions between phases as a function of temperature could be suitable. A phase transition indicates a change in the way the lipids are assembled and thus moves away from the ‘normal’ membrane. In this work, they used a technique called Electron Spin Echo (ESE) Spectroscopy.
ESE is a relatively new technique. It is described as a pulsed version of Electron Paramagnetic Resonance, which means that it detects orientational vibrations (librations) on the nanosecond time scale [1,2]. It does this only in a particular type of molecular species, one with an unpaired electron, called a free radical. Specialised molecular probes, called nitroxide labelled stearic acid, are used in these systems at low concentrations. In this work , it has given clear evidence that certain molecules (sucrose, trehalose) are beer cryoprotective agents than others (sorbitol).
What is not yet clear is what happens on thawing, or in more complicated systems. In order to understand the full process of freezing, or desiccation, on an organism, what happens during re-animation is also required. We also have yet to know what happens to membrane systems that comprise membrane proteins, like ion channels or hormone receptors. The tools for research in the direction of controlled desiccation/freezing, and re-animation, may be closer at hand. A membrane-form of trehalose is commercially available, called trehalose dibehenate (Figure, R). The fatty acid residues on this sugar mean that it will be thermodynamically favourable for this species to be located in the membrane. This guarantees that a number of trehalose molecules will be at the surface of the membrane, irrespective of the prevailing conditions. The presence of such a species on the membrane may confer stability during desiccation and freezing.
The exciting thing about work in this field is that we know the process happens frequently in nature, and that understanding it is the first step to harnessing it, probably with incalculable results.
 K. B. Konov, N. P. Isaev, S. A. Dzuba, J. Phys. Chem. B, 2014, 118, 12478. DOI: 10.1021/jp508312n
 N. V. Ivanisenko, S. A. Dzuba, Appl. Magn. Reson., 2013, 44, 883−891. DOI 10.1007/s00723-012-0436-4.
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