Collaborative Research: Novel Thermal Hysteresis Glycolipid Antifreeze in Insects and Plants

University Of Notre Dame, Notre Dame IN

Investigators

Abstract

PROJECT TITLE: Collaborative Research: Novel Thermal Hysteresis Glycolipid Antifreeze in Insects and Plants PRINCIPAL INVESTIGATOR: Duman, John G. PROJECT TITLE NUMBER: IOS 1025929 Organisms that are exposed to subzero temperature adapt by becoming either freeze tolerant (they survive being frozen) or they must become freeze avoiding to prevent freezing. Structurally diverse antifreeze proteins (AFPs) have evolved in many different organisms: animals, plants, bacteria, fungi, etc., but insect AFPs are arguably the most active. AFPs inhibit freezing by binding to the surface of ice crystals and/or ice nucleating surfaces, thereby reventing water molecules from joining the crystal surface. Consequently, AFPs lower the freezing point of an aqueous solution, but do not change the melting point, producing the thermal hysteresis (TH, difference between the freezing and melting points) characteristic of their presence. Previously, only antifreeze proteins were known to have this activity. Intellectual Merit. Recently, we identified novel glycolipids with TH equal to that of insect AFPs. These new antifreeze glycolipids (AFGLs) were found in several cold tolerant insects (both freeze tolerant and freeze avoiding), a frog, a fish, and a freeze tolerant plant. Since thermal hysteresis has previously been identified only in proteins, this novel discovery has the potential to transform our ideas on how organisms adapt to subzero temperatures. AFP function has been best studied in freeze avoiding species where they function to prevent freezing by blocking inoculative freezing across the surface from external ice and by inhibiting ice nucleators in body fluids. One species that we study, an Alaskan beetle, Cucujus clavipes, produces typical beetle type AFPs that assist them to deep supercool, so that they do not freeze even if taken to ?150oC. At ~-70oC the body water vitrifies, turns to glass, but does not freeze. C. clavipes is one of the species that produces AFGLs. We propose to continue studies of this interesting insect to determine the potential synergy in physiological function of these antifreezes. We will also investigate the structure and physiological function of AFGLs in two freeze tolerant insects, (Upis ceramboides) from Alaska (freeze tolerant to ~-60oC) and (Tipula trivittata) from Indiana (freeze tolerant to ~-28oC), and in a freeze tolerant plant, the bittersweet nightshade Solanum dulcamara from Indiana. The function of TH-antifreezes (AFPs or the AFGL) in freeze tolerant species is not well understood. Recall that these species have evolved to freeze and survive, so why have antifreeze? One possibility is that, since these organisms generally only survive freezing of their extracellular water, the AFGLs may function to prevent the lethal spread of ice from the extra- to the intra-cellular water. In fact, this may be the case since most of the AFGL in these species is associated with cell membranes, perfectly situated for this function. The primary scientific goal of this study is to (1) determine the structure of the AFGLs, and (2) to identify their physiological functions in both freeze tolerant and freeze avoiding organisms. The broader impacts of this study are three-fold: (1) potential cryopreservation of biomedical materials, (2) potential improved crop and horticultural plant cold tolerance and (3) a positive effect on biological education. AFPs, and now the novel AFGLs, have possible applications in the cryopreservation of cells, tissues and organs. AFGLs may provide freeze protection to cells, permitting them to be more easily freeze-preserved. AFGLs may also permit subzero storage of materials in the unfrozen state, mimicking their function in the deep supercooling C. clavipes from Alaska. There could also be applications in agriculture resulting in more cold tolerant plants. Students at the high school, undergraduate, PhD and post-doctoral levels will be directly involved in this study, thereby receiving interdisciplinary training ranging from field biology and physiological ecology to biochemistry and molecular biology. In addition, elucidation of these adaptations has the potential to attract new students and practicing scientists from diverse backgrounds. In this era of increased specialization when biochemists and ecologists, biologists and physicists or chemists seem to have few common interests, such studies can foster considerable interdisciplinary understanding and cooperation. Our recent initial publication of AFGLs has received widespread general attention, ranging from the NY Times to Nature magazine. Also, we will provide a service to cold tolerance researchers by screening their organisms for AFGLs.